Methods for evaluating tissue morphogenesis and activity

ABSTRACT

The present invention is based on the discovery that a true tissue morphogen such as OP-1 provided systemically, alone in its mature dimeric form, or as part of a soluble complex, can induce new replacement tissue regeneration at a localized, permissive defect site distal to the site of administration. Specifically, systemically administered protein is sufficient to induce formation of new functional replacement tissue, sufficient to repair a local defect in a tissue, including skeletal or orthopedic tissues, liver, pancreas, lung, cardiac, renal, uterine, intestinal, gastrointestinal _____ tissue. (As used herein, “orthopedic” or “skeletal” or “joint” or “chondrogenic” tissue is understood to encompass the skeletal and skeletal joint tissues: bone, cartilage, tendon, ligament, and synovial membrane tissues.) It further has been discovered that a single injection of morphogenic protein is sufficient to induce the desired biological effect, and that administration is not time-sensitive, provided mesenchymal progenitor cells are accessible to the defect site. That is, morphogenic protein can be provided to an individual having a local permissive defect site, shortly after creation of the defect, or at some significant time later, including, without limitation, after the initiation of fibrotic tissue formation. Thus, means now are available for enhancing restoration of tissue function and/or repair or regeneration of functional replacement tissue by systemically administering morphogenic protein, at times significantly after creation of the defect. The methods and formulations can be used to repair local defects without requiring surgical intervention; can enhance the rate and quality of new replacement tissue formation, particularly in compromised individuals with a reduced capacity to undergo spontaneous healing, and can be used to induce new tissue formation even after the initiation of fibrosis at the defect site. This discovery is disclosed in copending U.S. patent application (Attorney Docket CRP- 124, 2054/94 ) filed on even date herewith, the disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0001] The invention disclosed herein relates to materials and methodsfor inducing tissue-specific morphogenesis, and methods for evaluatingthe activity of morphogenic compounds.

BACKGROUND OF THE INVENTION

[0002] A class of proteins now has been identified that is competent toact as true tissue morphogens. That is, these proteins are able, ontheir own, to induce the migration, proliferation and differentiation ofprogenitor cells into functional replacement tissue. This class ofproteins, referred to herein as “osteogenic proteins” or “morphogenicproteins” or “morphogens,” includes members of the family of bonemorphogenetic proteins (BMPs) identified by their ability to induceectopic, endochondral bone morphogenesis. The morphogenic proteinsgenerally are classified in the art as a subgroup of the TGF-βsuperfamily of growth factors (Hogan (1996) Genes & Development10:1580-1594). Members of the morphogen family of proteins include themammalian osteogenic protein-1 (OP-1, also known as BMP-7, and theDrosophila homolog 60A), osteogenic protein-2 (OP-2, also known asBMP-8), osteogenic protein-3 (OP-3), BMP-2 (also known as BMP-2A orCBMP-2A, and the Drosophila homolog DPP), BMP-3, BMP-4 (also known asBMP-2B or CBMP-2B), BMP-5, BMP-6 and its murine homolog Vgr-1, BMP-9,BMP-10, BMP-11, BMP-12, GDF3 (also known as Vgr2), GDF8, GDF9, GDF10,GDF11, GDF12, BMP-13, BMP-14, BMP-15, GDF-5 (also known as CDMP-1 orMP52), GDF-6 (also known as CDMP-2), GDF-7 (also known as CDMP-3), theXenopus homolog Vg1 and NODAL, UNIVIN, SCREW, ADMP, and NEURAL.

[0003] Members of this family encode secreted polypeptide chains sharingcommon structural features, including processing from a precursor“pro-form” to yield a mature polypeptide chain competent to dimerize andcontaining a carboxy terminal active domain, of approximately 97-106amino acids. All members share a conserved pattern of cysteines in thisdomain and the active form of these proteins can be either adisulfide-bonded homodimer of a single family member or a heterodimer oftwo different members (see, e.g., Massague (1990) Annu. Rev. Cell Biol.6:597; Sampath, et al. (1990) J. Biol. Chem. 265:13198). See also, U.S.Pat. No. 5,011,691; U.S. Pat. No. 5,266,683, Ozkaynak et al. (1990) EMBOJ. 9:2085-2093, Wharton et al. (1991) PNAS 88:9214-9218), (Ozkaynak(1992) J. Biol. Chem. 267:25220-25227 and U.S. Pat. No. 5,266,683);(Celeste et al. (1991) PNAS 87:9843-9847); (Lyons et al. (1989 ) PNAS86:4554-4558). These disclosures describe the amino acid and DNAsequences, as well as the chemical and physical characteristics, ofthese osteogenic proteins. See also, Wozney et al. (1988) Science242:1528-1534); BMP 9 (WO93/00432, published Jan. 7, 1993); DPP (Padgettet al. (1987) Nature 325:81-84; and Vg-1 (Weeks (1987) Cell 51:861-867).

[0004] The morphogenic activities of these proteins allow them toinitiate and maintain the developmental cascade of tissue morphogenesisin an appropriate, morphogenically permissive environment, stimulatingstem cells to proliferate and differentiate in a tissue-specific manner,and inducing the progression of events that culminate in new tissueformation. These morphogenic activities also allow the proteins tostimulate the “redifferentiation” of cells previously induced to strayfrom their differentiation path. The proteins are useful in thereplacement of diseased or damaged tissue in a mammal, particularly whenthe damaged tissue interferes with normal tissue or organ function, suchas, for example, damaged lung tissue resulting from emphysema; cirrhotickidney or liver tissues; damaged heart or blood vessel tissue, as mayresult from cardiomyopathies and/or atherothrombotic or cardioembolicstrokes; damaged stomach tissue resulting from ulceric perforations ortheir repair; damaged neural tissue as may result form physical injury,degenerative diseases such as Alzheimer's disease or multiple sclerosisor strokes; damaged dentin and periodontal tissues as may result fromdisease or mechanical injury.

[0005] The proteins have been shown to have utility in repairing anumber of non-chondrogenic tissues, including dentin, liver, kidney,neural, cardiac lung, gastrointestinal tract tissue and the like. See,for example, W902/15323, published Sep. 17, 1992; W093/04692, publishedMar. 18, 1993; W094/06399, published Mar. 31, 1994; W094/03200,published Feb. 17, 1994; W094/06449, published Mar. 31, 1993;W094/06420, published Mar. 31, 1994. See also, U.S. Ser. Nos.08/404,113; 08/445,467; 08/432,883; 08/155,343; 08/260675; 08/445,468;08/461,397; 08/480,528; 08/402,542; 08/396,930; 08/751,227; (AttorneyDocket CRP 069 CP, filed Mar. 21, 1997) the disclosures of which areincorporated by reference.

[0006] Needs remain for compositions and methods for improved means forevaluating the in vivo activity and/or efficacy of these morphogenicproteins and analogs thereof It is anticipated that different morphogenswill have differing specific activities for effecting morphogenesis in agiven tissue or organ. It further is anticipated that analogs ofmorphogens, including candidate non-protein-based “small molecule”functional mimetics, will need to be evaluated for their ability tofunctionally substitute for a given morphogen in vivo. It further isanticipated that, for a given indication, such as treating an embolicstroke, for example, dosing and routes of administration can varydepending on the individual's overall health, age and condition. Thus,needs also remain for evaluating the pharmacokinetics of a morphogenicprotein or analog thereof, including evaluating dosing, preferredadministration times, and preferred administration routes foradministering a given morphogen, and/or analog to a given individual,for different therapeutic applications.

[0007] Accordingly, it is an object of the instant invention to provideformulations and methods of use thereof for quickly evaluating the invivo activity of morphogens and/or analogs thereof

[0008] These and other objects, along with advantages and features ofthe invention disclosed herein, will be apparent from the description,drawings and claims that follow.

SUMMARY OF THE INVENTION

[0009] The present invention is based on the discovery that a truetissue morphogen such as OP-1 provided systemically, alone in its maturedimeric form, or as part of a soluble complex, can induce newreplacement tissue regeneration at a localized, permissive defect sitedistal to the site of administration. Specifically, systemicallyadministered protein is sufficient to induce formation of new functionalreplacement tissue, sufficient to repair a local defect in a tissue,including skeletal or orthopedic tissues, liver, pancreas, lung,cardiac, renal, uterine, intestinal, gastrointestinal ____ tissue. (Asused herein, “orthopedic” or “skeletal” or “joint” or “chondrogenic”tissue is understood to encompass the skeletal and skeletal jointtissues: bone, cartilage, tendon, ligament, and synovial membranetissues.) It further has been discovered that a single injection ofmorphogenic protein is sufficient to induce the desired biologicaleffect, and that administration is not time-sensitive, providedmesenchymal progenitor cells are accessible to the defect site. That is,morphogenic protein can be provided to an individual having a localpermissive defect site, shortly after creation of the defect, or at somesignificant time later, including, without limitation, after theinitiation of fibrotic tissue formation. Thus, means now are availablefor enhancing restoration of tissue function and/or repair orregeneration of functional replacement tissue by systemicallyadministering morphogenic protein, at times significantly after creationof the defect. The methods and formulations can be used to repair localdefects without requiring surgical intervention; can enhance the rateand quality of new replacement tissue formation, particularly incompromised individuals with a reduced capacity to undergo spontaneoushealing, and can be used to induce new tissue formation even after theinitiation of fibrosis at the defect site. This discovery is disclosedin copending U.S. patent application (Attorney Docket CRP-124, 2054/94)filed on even date herewith, the disclosure of which is incorporatedherein by reference.

[0010] As disclosed herein, a method now is provided for effectivelyevaluating the in vivo activity of a given morphogen or morphogen analogin repairing a local tissue defect. The method comprises providing themorphogen or analog, as the case may be, systemically to an animalafflicted with a local tissue defect in need of repair. The morphogen oranalog can be provided by any systemic means, including orally,intravenously or intraperitoneally. The tissue can be any tissue in needof repair, including without limitation, any of the tissues describedherein. In addition, the methods provided herein allow one to evaluatereadily preferred formation compositions, including the value, if any,of added molecules such as targeting agents, antibiotics, analgesics,and the like; and to evaluate preferred binding agents, solutions, andother components adding value as administration route excipients.

[0011] The methods allow one to rapidly test any morphogen, includingany naturally-occurring or biosynthetic, e.g., genetically engineeredvariant thereof, including chimeras and muteins.

[0012] The methods also allows one to evaluate optional dosingstrategies, administration routes and/or administration times for agiven morphogen, morphogen analog, therapeutic indication and/orindividual condition. It is anticipated that these strategies will varyto some degree based on the type of defect, the type of tissue, thechoice of morphogen, and/or analog, and the condition of the individual.For example, aged individuals, or individuals having reduced blood flow,may require different dosing strategies and/or administration routes ascompared with younger individuals in good health. Testing dosingstrategies in these different patient populations allows one todetermine optimal molecules and conditions for administration.

[0013] The method provided herein also provides a reliable in vivo meansfor evaluating the efficacy of a candidate morphogen analog determinedto have utility in functionally mimicking a morphogen, as determined byone or more in vitro assays. In vitro assays for evaluating morphogen ormorphogen analog activity are described in numerous public sources,including WO 93/05751, published Apr. 1, 1993, as well as in U.S. Ser.No. 08/432,883, filed May 2, 1995, and U.S. Ser. No. 08/727,118, filedOct. 8, 1996, the disclosures of which are incorporated herein byreference. The methods also provide ready and reliable means fordetermining what, if any, may be the toxicity level of a given candidateanalog.

[0014] The assay involves creating a local defect in a tissue ofinterest and administering the morphogen or analog systemically. In oneembodiment, a biocompatible, biodegradable matrix is implanted at asubcutaneous site and the morphogen or analog is administeredsystemically, for example, interperitoneally on intravenously. In oneembodiment, the matrix is a bone-derived collagen matrix and activemorphogens or analogs are competent to induce new cartilage and boneformation at the collagen implant site, which can be evaluated bystandard histology 12 days post implant. In another embodiment, apermissive local defect site is created in existing tissue.

[0015] As contemplated herein, a “permissive” site is a local site of atissue defect in need of repair and to which progenitor cells areaccessible. Mesenchymal progenitor cells typically become available to adefect locus at least by 6-24 hours post trauma as part of theinflammatory response triggered by the initial trauma. Specifically,these progenitor cells (stem cells) are recruited to the site by thechemokines and growth factors activated by the inflammatory response.These recruited progenitor cells form a condensed mass at the defectlocus, typically referred to as a callus, and are available todifferentiate into a specific tissue type in response to locallyavailable, specific, tissue-inductive signals. In the absence of suchtissue-inductive signals, these progenitor cells typically are inducedto differentiate into fibroblasts by the chemokines and growth factors(e.g., PDGF, TGFβ, IL-1 and the like). The committed fibroblasts thenare competent to generate a non-specific extracellular matrixcharacteristic of fibrotic “scarring” tissue and which can be resorbedover time. Such scarring is characteristic of cirrhotic tissue or tissueinfarcts, as can occur in lung, liver, kidney, and cardiac tissues, forexample.

[0016] In another embodiment, a local defect site is created in a tissueof interest, such as lung, cardiac, pancreas, liver, gastrointestinaltract, and neural tissue, for example. The methods and compositions arecontemplated to assist in evaluating preferred administration protocolsfor repairing and/or restoring function to tissues such as skeletaltissues (including bone, cartilage, ligament, tendon and synovialmembrane tissues), and liver, kidney, lung, pancreas, spleen, uterine,cardiac, thyroid, gastrointestinal tract, neural tissues, sense organs,and the like.

[0017] In one preferred embodiment, the assay can be used to evaluateoptimal administration times. For example, in one embodiment, theprotein or candidate analog is provided at least 6 hours post trauma, or10-24 hours post trauma. In another embodiment, protein or analog isprovided systemically any time between 24-36 hrs and/or between 36-72hrs, and/or between 72-120 hrs, and/or between 120-168 hrs post-trauma.In another embodiment, the assay is used to evaluate optimaladministration routes, times, and dosages for promoting or inducingtissue repair under refractory healing conditions. As used herein,“refractory healing” refers to any defect where, due to the nature ofthe defect or the condition of the individual (aged, obese, smoker,diabetic, steroidal user), for example, spontaneous formation of newreplacement tissue sufficient to correct the defect does not occur.

[0018] In another aspect, the instant invention provides methods forassessing the ability of a morphogen or morphogen analog to regeneratelost or damaged tissue in vivo in an existing tissue or organ. Inanother aspect, the invention provides methods for assessing the abilityor a morphogen or analog to maintain normal tissue function followingtissue injury, or in anticipation of such injury. As disclosed herein,methods of repair include treatment of both closed and open defects.Examples of defects include, but are not limited to, tissue defects.

[0019] In another aspect, the instant invention provides a kit forpractice of the above-described methods. As contemplated herein, oneembodiment of a kit includes a collagen matrix implant material and aformulation of morphogen and/or analog for systemic administration. Inanother embodiment, the kit comprises the morphogenic protein or analogand systemic administration carrier (e.g., a liquid carrier) arepackaged in the same receptacle. In other embodiments, the morphogenicprotein or analog is provided in lyophilized form and reconstituted in agiven carrier, e.g., aqueous buffer, in the same receptacle.

[0020] Exemplary formulations for testing using the methods of theinvention include providing the protein or analog as a liquidformulation administered intravenously. In another embodiment, theprotein or analog is provided in a liquid formulation intraperitoneally.In still another embodiment, the protein or analog is provided in liquidor tablet or other non-liquid form for oral administration, includingdisposed in biocompatible, biodegradable or bioerodible microspheres andother delivery vehicles, or otherwise combined with suitable bindingagents as described herein. Another preferred embodiment can have a drypowder configuration that is solubilized just prior to administration.One suitable formulation results from first dispersing morphogenicprotein or analog in a liquid carrier such as water with or withoutexcipient, followed by lyophilization. In one formulation tested, thecomposition is a solution made by combining the protein together with anacidic buffered solution, e.g., pH 4.0-4.5, for example an acetate orcitrate buffer. Still another formulation is a suspension formed bydisbursing osteogenic protein in a physiologically buffered solution,such as phosphate buffered saline (PBS).

[0021] As contemplated herein, morphogenic proteins useful forevaluating in the methods of the invention include, but are not limitedto, OP-1, OP-2, BMP-2, BMP-4, BMP-5 and BMP-6. A currently preferredmorphogenic protein is OP-1. As used herein, the terms “morphogen”,“bone morphogen”, “bone morphogenic protein”, “BMP”, “osteogenicprotein” and “osteogenic factor” embrace the class of proteins typifiedby human osteogenic protein 1 (hOP-1). Nucleotide and amino acidsequences for hOP-1 are provided in Seq. ID Nos. 1 and 2, respectively.For ease of description, hOP-1 is recited herein below as arepresentative morphogenic protein. It will be appreciated by theartisan of ordinary skill in the art, however, that OP-1 merely isrepresentative of the TGF-β subclass of true tissue morphogens, and isnot intended to limit the description. Other known, and useful proteinsinclude, BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9,BMP-10, BMP-11, BMP-12, BMP-13, BMP-15, GDF-1, GDF-2, GDF-3, GDF-5,GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, NODAL, UNIVIN,SCREW, ADMP, NEURAL and morphogenically active amino acid variantsthereof In one preferred embodiment, the proteins useful in theinvention include biologically active species variants of any of theseproteins, including conservative amino acid sequence variants, proteinsencoded by degenerate nucleotide sequence variants, and osteogenicallyactive proteins sharing the conserved seven cysteine skeleton as definedherein and encoded by a DNA sequence competent to hybridize to a DNAsequence encoding a morphogenic protein disclosed herein, including,without limitation, OP-1, BMP-5, BMP-6, BMP-2, BMP-4 or GDF-5, GDF-6 orGDF-7. In another embodiment, useful proteins include those sharing theconserved seven cysteine domain and sharing at least 70% amino acidsequence homology (similarity) within the C-terminal active domain, asdefined herein. In another embodiment, useful proteins include thosesharing greater than 60% identity in the C-terminal domain. In stillanother embodiment, useful osteogenic proteins can be defined asosteogenically active proteins having any one of the generic sequencesdefined herein, including OPX (SEQ ID No:3) and Generic Sequences 7 and8 (Seq. ID Nos.4 and 5), or Generic Sequences 9 and 10 (Seq. ID Nos.6and 7).

[0022] As contemplated herein, the methods of the invention are usefulfor evaluating morphogenic properties of a morphogen analog, e.g., anycandidate compound competent to induce a morphogen-mediated biologicaleffect. Morphogen analogs include homologs and ligand analogs that cansubstitute for a morphogen in a ligand-morphogen receptor bindingintraction, as well as functional mimetics competent to inducebiological effect of morphogenesis by inducing a downstream effectnormally stimulated by ligand-morphogen receptor binding under nativeconditions.

[0023] As a result of the present analog identification methods, theinvention provides means for identifying and producing therapeutic-grademorphogen analogs. The invention further provides for identifying andproducing a derivative of a candidate morphogen analog in which anyundesirable properties of the initially identified analog, such as invivo toxicity or a tendency to degrade upon storage, are mitigated.

[0024] Still another embodiment contemplates assay methods useful indetermining proper morphogen or analog dosing and/or progression ofmorphogenesis.

[0025] In any treatment method of the invention, “administration ofmorphogenic protein or analog” refers to the administration of theprotein or an analog thereof, either alone or in combination with othermolecules. For example, the mature form of the morphogen may be providedin association with its precursor “pro” domain, which is known toenhance the solubility of the protein. As used herein, “soluble form” ofa morphogenic protein is understood to mean the dimeric speciescomplexed with part or all of a morphogenic protein pro domain. See, forexample, WO94/03600, published Feb. 18, 1994 and/or Jones et al., (1994)Growth Factors 11: 215-225, for a detailed description of the solublecomplex form of morphogenic proteins. Other useful molecules known toenhance protein solubility include casein and other milk components, aswell as various serum proteins.

[0026] The foregoing and other objects, features and advantages of thepresent invention will be made more apparent from the following detaileddescription of the invention.

DESCRIPTION OF THE DRAWINGS

[0027] FIGS. 1A-1C is a graph showing bone forming activity induced bysystemic OP-1.

[0028] FIGS. 2A-2C is a graph showing the effect of timing of OP-1administration on bone forming activity. Bovine collagen carrier (25 mg)was implanted at intramuscular and subcutaneous sites, and OP-1 wasadministered via the tail vein (1500 ug as a single injection) 1, 3, 5,7, or 9 days after collagen implantation. Bone-forming activity wasdetermined by histology, alkaline phosphatase, and calcium content at 12days after administration of OP-1.

[0029]FIGS. 3A and 3B show the effect of the age of an animal onbone-forming activity induced by systemically-administered OP-1. Bovinecollagen carrier (25 mg) was implanted subuctaneously andintramuscularly. OP-1 was administered via the tail vein (2500 ug as asingle injection 24 hours after carrier implantation. Bone-formingactivity was determined by alkaline phosphatase activity (FIG. 3A),calcium content (FIG. 3B), and by histology conducted on implants at 12days after OP-1 implantation.

[0030]FIG. 4 is a table showing the percent homology of variousmorphogens to human OP-1 c-terminal 7-cysteine region.

DETAILED DESCRIPTION

[0031] An assay method now has been discovered for evaluating theefficacy and/or pharmacokinetic properties of morphogenic proteins andanalogs thereof. The method comprises the steps of systemicallyadministering a morphogenic protein or an analog thereof in an animalhaving a local permissive defect site and evaluating the ability of theprotein or analog administered, under the conditions of the assay, toinduce formation of functional replacement tissue at the defect site.The method does not require inclusion of an exogenous matrix material orthe need to provide the protein or analog directly to the defect locus.A local permissive defect site readily can be created by implanting amatrix material in a intramuscular or subcutaneous site in an animal,such as a rat. In one embodiment, the material is a demineralized,deproteinated collagen matrix. In another embodiment, the matrix is anyother biocompatable, biodegradable, biologically inert scaffoldingmaterial, preferably porous and substantially acellular. In stillanother embodiment, a permissive local defect site is created in anexisting tissue. For example, a fracture can be induced in bone, and atear or chondral defect can be induced in cartilage. Similar mechanicalor toxin-induced defects can be induced in lung, cardiac, liver,pancreatic, uterine and other tissues, to name but a few.

[0032] The defects also can be created in standard, well characterizedanimal models representative of different patient populations, as ameans for evaluating morphogen or analog efficacy and/orpharmacokinetics in different therapeutic conditions. Examplary patientpopulations include, without limitation, juveniles, aged, diabetic,hypertensive, obese, immune-comprised animals, and the like.

[0033] Provided below are detailed descriptions of suitable morphogenicproteins and analogs, and formulations useful in the methods,compositions and assays of this invention, as well as methods for theiradministration and application; and numerous, nonlimiting exampleswhich 1) illustrate the suitability of the morphogenic proteins,analogs, formulations, methods and assays described herein; and 2)provide assays with which to test candidate proteins and analogs,formulations for their efficacy in different tissues, for repairingvarious defects, and for measuring efficacy and/or pharmacokinetics indifferent patient populations.

[0034] In order to more clearly and concisely describe the subjectmatter of the claimed invention, the following definitions are intendedto provide guidance as to the meaning of specific terms used in thewritten description and appended claims.

[0035] As embodied herein, the expression “maintaining normal tissuefunction” means both regaining or restoring tissue function lost due toan injury or acquired or congenital defect, as well as protecting thetissue at risk of damage from injury. Restoring tissue function caninclude regenerating new tissue and/or simulating existingdifferentiated tissue cells to continue expressing their phenotype as inthe case of senescent cells. “Depressed tissue function” level refers toa diminished to deficient tissue function as a result of a tissue injuryor disease. The expression “enhance viability of” a tissue or organ, asused herein, means protection from, reduction of and/or elimination ofreduced or lost tissue or organ function as a result of tissue necrosisand/or fibrosis, particularly immune response-mediated tissue necrosisand/or fibrosis. “Alleviating” means protection from, reduction ofand/or elimination of, undesired tissue destruction. “Transplanted”living tissue includes both tissue grafts and cellular transplants, asin the case of transplanted isolated progenitor or stem cells, forexample, which may be implanted alone or in association with a temporaryscaffolding. Tissues may be autologous or allogenic tissue and/orsynthetic tissue created, for example, by culturing hepatic cells in thepresence of an artificial matrix. “Morphogenically permissiveenvironment” is understood to mean an environment competent to allowtissue morphogenesis to occur. Finally, “symptom alleviating cofactor”refers to one or more pharmaceuticals which may be administered togetherwith the therapeutic agents of this invention and which alleviate ormitigate one or more of the symptoms typically associated with thetissue injury and/or tissue function loss. Exemplary cofactors includeantibiotics, antiseptics, non-steroidal anti-inflammatory agents, andthe like.

[0036] “Defect” or “defect site” or “defect locus”, as contemplatedherein, can define any structural disruption in a tissue or organrequiring repair. Systemically administered morphogenic protein canenhance the rate of recruiting, proliferating and differentiatingmesenchymal progenitor cells (stem cells). Repair of such tissue defectsis dependent on the presence of available or accessible mesenchymalprogenitor cells.

[0037] “Repair” is intended to mean formation of new tissue which issufficient to restore function and/or otherwise functionally correct adefect in a mammal. Repair does not, however, mean, or otherwisenecessitate, a process of complete healing or a treatment which is 100%effective at restoring a defect to its pre-defectphysiological/structural/mechanical state.

[0038] In addition to morphogenic proteins, various systemic factors,hormones, enzymes, enzyme inhibitors and/or chemoattractant/chemotacticfactors, therapeutic compositions, antibiotics, or other bioactiveagents also can be contained within formulation for use in theinvention. Thus, various known growth factors such as EGF, PDGF, IGF,FGF, TGF-a, and TGF-β can be combined with a morphogenic formulationdescribed herein and administered systemically.

[0039] “Morphogen”, “morphogenic protein”, “osteogenic protein”, or“bone morphogenic protein,” generally is understood to mean a proteinwhich can induce the full cascade of morphogenic events culminating innew organ-specified tissue formation. As described elsewhere herein, theclass of proteins is typified by human osteogenic protein (hOP1). Otherosteogenic proteins useful in the practice of the invention includeosteogenically active forms of OP1, OP2, OP3, BMP2, BMP3, BMP4, BMP5,BMP6, BMP9, DPP, Vg1, Vgr, 60A protein, GDF-1, GDF-3, GDF-5, 6, 7,BMP10, BMP11, BMP12, BMP13, BMP15, UNIVIN, NODAL, SCREW, ADMP or NEURALand amino acid sequence variants thereof. In one currently preferredembodiment, osteogenic protein includes any one of: OP1, OP2, OP3, BMP2,BMP4, BMP5, BMP6, BMP9, and amino acid sequence variants and homologsthereof, including species homologs thereof Particularly preferredproteins are those comprising an amino acid sequence having at least 70%homology with the C-terminal 102-106 amino acids, defining the conservedseven cysteine domain, of human OP-1, BMP2, and related proteins.Certain preferred embodiments of the instant invention comprise theosteogenic protein, OP-1 and proteins sharing greater than 60% aminoacid sequence identity with OP-1 in the C-terminal seven cysteinedomain. Certain other preferred embodiments comprise mature OP-1solubilized in a physiological saline solution. As further describedelsewhere herein, the proteins suitable for use with Applicants'invention can be identified by means of routine experimentation usingthe art-recognized bioassay described by Reddi and Sampath. A detaileddescription of useful morphogenic proteins is provided below.

[0040] In general terms, an “analog” is understood to be a functionalequivalent of a given substance and can be a substitute for saidsubstance, including as a therapeutic substitute. An analog also can bea structural equivalent. As used herein, a “morphogen analog” is asubstance that mimics a biological effect induced and/or mediated by amorphogen, such as OP-1. Any substance having such mimetic properties,regardless of the chemical or biochemical nature thereof, can be used asa morphogen analog herein. A morphogen analog as contemplated herein canbe a simple or complex substance produced by a living system or throughchemical or biochemical synthetic techniques. It can be a substance thatoccurs in nature or it can be a novel substance, e.g., preparedaccording to principles of rational drug design. It can be a substancethat structurally resembles a solvent-exposed morphogen surface epitopeimplicated in receptor interactions, a substance that otherwisestimulates a morphogen receptor displayed on the surface of a morphogenresponsive cell, or a cell-membrane permanent substance or otherwiseintracellular-acting molecule that interacts with an intracellularcomponent of the signal transduction machinery of a morphogen-responsivecell and thereby stimulates a morphogen specific biological effect. Suchintracellular acting morphogen analogs also are referred to herein as“downstream morphogenesis inducers”. As used herein, a morphogen analogcan be referred to as a “mimic” or a “mimetic”.

[0041] In another embodiment, the morphogen analog useful in the presentinvention comprises a candidate compound or an agent which acts as anagonist of a morphogen receptor. An “agonist” of a receptor means acompound which binds to the receptor and for which such binding has asimilar functional result as binding of a morphogen to the receptor.That is, the compound upon interaction with the receptor, produces thesame or a substantially similar transmembrane and/or intracellulareffect as a morphogen. Thus, an agonist of a morphogen receptor binds tothe receptor and such binding has the same or a similar functionalresult as morphogen binding (e.g., induction of morphogenesis). Theactivity or potency of an agonist can be less than that of the naturalmorphogen, in which case the agonist is said to be a “partial agonist,”or it can be equal to or greater than that of the natural ligand, inwhich case it is said to be a “full agonist.” Thus, for example, a smallpeptide or other molecule which can mimic the activity of a morphogen inbinding to and activating the morphogen's receptor can be employed as anequivalent of the morphogen. Preferably the agonist is a full agonist,but partial morphogen receptor agonists can also be advantageouslyemployed. Methods of identifying such agonists are disclosed herein andinclude assays for compounds which induce morphogen-mediated responses(e.g., induction of differentiation of metanephric mesenchyme, inductionof endochondral bone formation, and the like). Such an agonist also canbe referred to as a morphogen “mimic,” “mimetic,” or “analog.”

[0042] Also by way of example and without being limited hereto, anothertype of morphogen analog usefull in the present invention can beprepared through judicious application of the principles of biosyntheticantibody binding site (BABS) technology as set forth in U.S. Pat. Nos.5,132,405, 5,091,513 and 5,258,498, the teachings of which areincorporated herein by reference. BABS analog constructs can be preparedfrom antibodies, preferably produced by hybridoma cells, that bindspecifically to a morphogen cell surface receptor. Alternatively, BABSanalysis can be prepared from anti-idiotypic antibodies specificallyreactive with the antigen binding site of an antibody that blocksmorphogen biological activity. Vukicevic et al. (1994) Biochem. Biophys.Res. Comm. 198:693-700 teaches the preparation of OP-1 specificmonoclonal antibodies. Skilled artisans will appreciate that suchantibodies can be used as immunogens in the routine preparation ofanti-idiotypic antibodies from which BABS analogs of the presentinvention can be prepared.

[0043] A structurally distinct class of morphogen analogs, again setforth herein for illustration and not for limitation, can be preparedthrough application of the principles of directed molecular evolution asset forth in Tuerk et al. (1990) Science 249:505-510, Famulok et al.(1992) Angew. Chem. Intl. Ed. Engl. 31:979-988 and Bock et al. (1992)Nature 355:564-556, the teachings of each of which are incorporated byreference herein. The directed molecular evolution process involvesisolation of a nucleic acid molecule, typically an RNA, that binds withhigh affinity to a selected ligand such as a protein. Such a nucleicacid molecule is referred to in the art as an “aptamer.” The desiredaptamer is initially present in a random pool of nucleic acid molecules,and is isolated by performing several rounds of ligand-affinity basedchromatography alternating with PCR-based amplification ofligand-binding nucleic acids. Bock et al. (1992), above, havedemonstrated the preparations of aptamers, suitable for in vivo use inmammals, that specifically inhibit the blood clot promoting factor,thrombin. As contemplated herein, such aptamers can be derived from amorphogen.

[0044] Yet another structurally distinct class of morphogen analogs canbe prepared by selecting appropriate members of a random peptide library(Scott et al. (1990) Science 249:386-390) or a combinatoriallysynthesized random library of organic or inorganic compounds (Needels etal. (1993) Proc. Natl. Acad. Sci. USA 90:10700-10704; Ohlmeyer et al.(1993) Proc. Natl. Acad. Sci. USA 90:10922-10926). Skilled artisans willappreciate that the foregoing and other related technologies, takentogether with long-established principles of screeningbiologically-produced substances, offer a wide array of candidatesubstances for screening for morphogen analog activity. As will beappreciated by the skilled artisan, the product of such a library screencan mimic OP-1 or another morphogen as a ligand for morphogen receptorbinding. Alternatively, the product can induce a morphogen-specificbiological effect through one or more intracellular interactions. Thus,a naturally-sourced or genetically engineered OP-1 or other morphogenanalog, morphogen receptor analog or biological functional mimetic, cancomprise a polypeptide, polynucleotide, carbohydrate, lipid, amino acid,nucleic acid, sugar, fatty acid, steroid, or a derivative of any one ofthe aforementioned compounds. It can be an intermediate or end productof metabolism of a eukaryotic or prokaryotic cell. Alternatively, theanalog can be a biological response modifier or a toxin. Finally, theanalog can be a molecule competent to induce expression of an endogenousmorphogen.

[0045] “Binding Agent”, as used herein, means anyphysiologically-compatible material which, when admixed with amorphogenic protein as defined herein, enhances a desired physicalproperty of the formulation without substantially destroying thebiological activity of the protein in vivo. Binding agents arecontemplated to have utility when oral administration is desired. Amongthe other characteristics of a preferred binding agent is an ability torender the device: pliable, shapeable and/or malleable. Additionally, incertain preferred embodiments, a binding agent can achieve theaforementioned features and benefits when present in low proportions.

[0046] Those binding agents contemplated as useful herein include, butare not limited to: art-recognized suspending agents,viscosity-producing agents and emulsifying agents. In particular,art-recognized agents, such as cellulose gum derivatives, sodiumalginate, and gelatin powder can be used. More particularly, cellulosicagents such as alkylcelluloses, are preferred including agents such asmethylcellulose, methylhydroxyethylcellulose, hydroxyethylcellulose,hydroxypropylmethylcellulose, carboxymethylcellulose, sodiumcarboxymethylcellulose, and hydroxyalkylcelluloses, to name but a few.Currently, the most preferred is carboxymethylcellulose, including thesodium salt thereof. Other useful binding agents include, but are notlimited to, dextran, mannitol, white petrolatum, sesame oil andadmixtures thereof. In view of the teachings set forth herein, theartisan can identify suitable equivalents of the above-identifiedbinding agents using merely routine experimentation and ordinary skill.

[0047] “Wetting Agent” or “carrier”, as used herein, means anyphysiologically-compatible aqueous solution, provided it does notinterfere with the in vivo biological activity of the osteogenicprotein. Currently preferred wetting or carrier agents include aqueoussolutions competent to solubilize or otherwise suspend the protein insolution such that it can be administered in liquid form to anindividual. Currently preferred carriers include, without limitation,physiological saline, phosphate buffered saline (PBS), acetate bufferedsolutions (pH4.5) and the like. Equivalents can be identified by theartisan using no more than routine experimentation and ordinary skill.

[0048] Exemplary Tissues:

[0049] Some exemplary tissues and the etiologies of various defectstreatable by the methods and compositions described herein and enabledby this disclosure, and useful in the evaluative assays enabled by thisdisclosure, are listed below. It will be understood by those skilled inthe art that the recitation is not intended to be limiting in anyway. Inaddition to the tissues recited herein, other tissues, including,without limitation, pancreas, uterine, ovarian, gastrointestinal tract,colon, intestinal, dermal, and periodontal tissues are treatable by themethods and compositions described herein.

[0050] Cardiac Tissue:

[0051] Adult mammalian cardiac muscle has extremely limited powers ofgrowth and regeneration. As a result, damage or loss of myocardium due,for example, to myocardial infarction, congestive heart failure,physical trauma (e.g., in an automobile accident), or infection,typically results in a permanent and often progressive loss offunctional myocardium.

[0052] Subjects that can benefit from the methods and compositions ofthe invention include individuals at risk of, or afflicted with, loss ofor damage to myocardium. Such subjects include subjects alreadyafflicted with the loss of myocardial tissue, such as those which havealready suffered a myocardial infarction, physical trauma to the heart(e.g., in an automobile accident), or those already suffering fromcongestive heart failure, as well as subjects reasonably expected tosuffer from myocardial infarction or congestive heart failure.

[0053] The methods and compositions are competent to induce a process ofproliferation and/or differentiation of progenitor stem cells at a siteof lost or damaged mammalian myocardium to produce new and functionalmammalian myocardium, thereby restoring or regenerating the lost ordamaged tissue in whole or in part. The treatment also can be used tocorrect chronically deteriorating mammalian myocardium (e.g., due tocongestive heart failure or chronic myopathy). In one embodiment, asubject that has already suffered from one or more myocardial infarctscan undergo surgery to remove scar tissue, and morphogen or analog canbe administered systemically to induce cardiac tissue morphogenesis.

[0054] Neural Tissue:

[0055] Like cardiac tissue, mammalian neural tissue has extremelylimited powers of growth and regeneration. The morphogen or analog canbe used in the methods and compositions of the present invention uponinjury to a neural pathway, or in anticipation of such injury, for atime and at a concentration sufficient to maintain the neural pathway,including repairing damaged pathways, or inhibiting additional damagethereto.

[0056] In particular, the morphogens and analogs can be used to repairdamaged pathways, including transected or otherwise damaged nerve fibers(nerves) requiring regeneration of neuronal processes, particularlyaxons, over extended distances to bridge a gap in the nerve itself, orbetween the nerve and a post-synaptic cell. Specifically, the morphogensand analogs described herein are capable of stimulating complete axonalnerve regeneration, including vascularization and reformation of theprotective myelin sheath. They also are competent to form functionalreplacement neural pathways in the central nervous system, such as inthe repair of damaged or detached retinas, or other damage to the opticnerve.

[0057] The morphogens and analogs also are useful for enhancing survivalof neuronal cells at risk of dying, thereby preventing, limiting orotherwise inhibiting damage to neural pathways. Non-mitotic neurons areat risk of dying as a result of a neuropathy or other cellulardysfunction of a neuron or glial cell inducing cell death, or followinga chemical or mechanical lesion to the cell or its surrounding tissue.The chemical lesions may result from known toxic agents, including lead,ethanol, ammonia, formaldehyde and many other organic solvents, as wellas the toxins in cigarette smoke and opiates. Excitatory amino acids,such as glutamate also may play a role in the pathogenesis of neuronalcell death (see Freese et al. (1990) Brain Res. 521:254-264). Neuronalcell death also is thought to be a significant contributing factor in anumber of neurodegenerative diseases, including Alzheimer's disease,Huntington's chorea, and Parkinson's disease, amyotrophic lateralsclerosis and multiple sclerosis. The etiology of these neuropathies maybe metabolic, as results in hepatic encephalopathy, infectious, toxic,autoimmune, nutritional or ischemic. In addition, ethanol and a numberof other toxins also have been identified as significant contributingfactors in neurodegenerative diseases.

[0058] The morphogens and analogs described herein also are useful forproviding neuroprotective effects to alleviate neural pathway damageassociated with the body's immune/inflammatory response to an initialinjury to nerve tissue. Such a response may follow trauma to nervetissue, caused, for example, by an autoimmune dysfunction, neoplasticlesion, infection, chemical or mechanical trauma, disease, byinterruption of blood flow to the neurons or glial cells, for examplefollowing ischemia or hypoxia, or by other trauma to the nerve orsurrounding material. For example, the primary damage resulting fromhypoxia or ischemia-reperfusion following occlusion of a neural bloodsupply, as in an embolic stroke, is believed to be immunologicallyassociated. In addition, at least part of the damage associated with anumber of primary brain tumors also appears to be immunologicallyrelated. Providing the morphogen to the mammal systemically, forexample, intravenously or indirectly by oral administration, may be usedto alleviate and/or inhibit the immunologically related response to aneural injury. Where the injury is to be induced, as during surgery orother aggressive clinical treatment, the morphogen or agent may beprovided prior to induction of the injury to provide a neuroprotectiveeffect to the nerve tissue at risk.

[0059] In still another aspect, the invention described herein providesmethods for evaluating the efficacy and/or pharmacokinetics ofmorphogens and analogs competent to support the growth and maintenanceof differentiated neurons, including inducing neurons to continueexpressing their phenotype. This activity can be used in the treatmentof nerve tissue disorders where loss of function is caused by reduced orlost cellular metabolic function and cells become senescent orquiescent, such as is thought to occur in aging cells and to bemanifested in Alzheimer's disease. Providing morphogen systemically byparenteral or oral administration stimulates these cells to continueexpressing their phenotype, significantly inhibiting and/or reversingthe effects of the cellular metabolic dysfunction, thereby maintainingthe neural pathway at risk.

[0060] The invention also can be used for evaluating morphogens, analogsand their pharmacokinetics in treating traumatic injuries to the centralnervous system that are caused by mechanical forces, such as a blow tothe head. Trauma can involve a tissue insult selected from abrasion,incision, contusion, puncture, compression, etc., such as can arise fromtraumatic contact of a foreign object with any locus of or appurtenantto the mammalian head, neck or vertebral column. Other forms oftraumatic injury can arise from constriction or compression of mammalianCNS tissue by an inappropriate accumulation of fluid (e.g., a blockadeor dysfunction of normal cerebrospinal fluid or vitreous humor fluidproduction, turnover or volume regulation, or a subdural or intracranialhematoma or edema). Similarly, traumatic constriction or compression canarise from the presence of a mass of abnormal tissue, such as ametastatic or primary tumor.

[0061] Liver:

[0062] Unlike most other organs in the body the liver has a definedregenerative capacity following hepatic tissue damage or cell death.Specifically, while hepatocytes do not proliferate actively followingfetal and post natal liver growth, normally quiescent hepatocytes dodivide in response to cell death or loss of liver tissue. However, wheretissue damage is extensive and/or chronic, permanent tissue damage canresult, reducing the organ's viability and functional capacity.Permanent hepatic tissue damage typically is characterized by extensivenecrosis and/or fibrogenesis or scarring (cirrhosis). Another source ofnonreparative damage results from hepatic neoplasms and metastaticcarcinomas.

[0063] Where either the mass of liver cells is sufficiently diminishedor their function sufficiently impaired, hepatic failure ensues. Theetiology of hepatic failure may be metabolic (e.g., altered bilirubinmetabolism or fatty acid storage), infectious (e.g., induced by viralhepatitis, hepatic schistomiasis, syphilis, or ascariaris), toxic (e.g.,induced by ethanol, ammonia, phenol, and other environmental toxins,fatty acids, drugs and/or their metabolites), autoimmune, ischemic ornutritional (e.g., alcoholic liver disease liver failure also ischaracterized by severe and often life-threatening bleeding, due to thereduced production of essential blood clotting factors). Hepatic failurealso can induce neurological dysfunction, characterized broadly ashepatic encephalopathy, as well as associated renal failure, jaundice,pulmonary complications, and a host of disorders associated withhormonal imbalances.

[0064] Gastrointestinal Tract

[0065] The methods and compositions of the invention are useful forevaluating morphogens and analogs in protecting the luminal lining ofthe gastrointestinal tract from ulceration, particularly in individualsat risk for ulcer formation. Specifically, the morphogens and analogsdescribed herein can be assessed for their efficacy in limiting theproliferation of epithelial cells, inhibiting the inflammation normallyassociated with ulcerative disease, inhibiting scar tissue formation,and inducing repair and regeneration of the ulcerated tissue.

[0066] In one aspect, the invention features compositions and methodsfor evaluating optimal therapeutically effective amounts of amorphogenic protein or analog sufficient to maintain the integrity ofthe GI tract luminal lining that comprise the step of systemicallyadministering to a mammal upon injury to all or a portion of the GItract luminal lining or in anticipation of such injury and evaluatingthe repair of the ulcerated tissue, and/or inhibition of damage thereto.

[0067] In one preferred embodiment of the invention, the ulcers createdfor assay and/or treatable according to the invention include thosefound in the ileum which cause regional ileitis, those found in thecolon which cause ulcerative colitis, regional enteritis (Crohn'sdisease), proctitis and other forms of inflammatory bowel disease (IBD),gastric ulcers such as those found in the stomach, small intestines,duodenum and esophagus; and ulcers found in the mouth. The compositionsand methods described herein are particularly useful in treatingmucositis lesions caused by chemotherapy or radiation therapy.

[0068] Lung:

[0069] A variety of lung diseases are characterized by airwayinflammation, including chronic bronchitis, emphysema, idiopathicpulmonary fibrosis and asthma. Another type of lung-related inflammationdisorders are inflammatory diseases characterized by a generalized,wide-spread, acute inflammatory response such as adult respiratorydistress syndrome. Another dysfunction associated with the inflammatoryresponse is that mounted in response to injury caused by hyperoxia,e.g., prolonged exposure to lethally high concentrations of O₂ (95-100%O₂). Similarly, reduced blood flow to a tissue (and, therefore reducedor lack of oxygen to tissues), as described below, also can induce aprimary tissue injury that stimulates the inflammatory response.

[0070] The assays of the present invention are competent to evaluateefficacy and/or pharmacokinetics of morphogens and analogs in restoringlung tissue function and/or tissue loss due to these and other sourcesof lung tissue damage.

[0071] The means for making and using the formulations and methods ofthe invention, as well as other material aspects concerning their natureand utility, including how to make and how to use the subject matterclaimed, will be further understood from the following, whichconstitutes the best mode currently contemplated for practicing theinvention. It will be appreciated that the invention is not limited tosuch exemplary work or to the specific details set forth in theseexamples.

I. PROTEIN CONSIDERATIONS

[0072] A. Biochemical Structural and Functional Properties of BoneMorphogenic Proteins

[0073] In its mature, native form, natural-sourced morphogenic proteinis a glycosylated dimer typically having an apparent molecular weight ofabout 30-36 kDa as determined by SDS-PAGE. When reduced, the 30 kDaprotein gives rise to two glycosylated peptide subunits having apparentmolecular weights of about 16 kDa and 18 kDa. In the reduced state, theprotein has no detectable osteogenic activity. The unglycosylatedprotein, which also has osteogenic activity, has an apparent molecularweight of about 27 kDa. When reduced, the 27 kDa protein gives rise totwo unglycosylated polypeptide chains, having molecular weights of about14 kDa to 16 kDa. Typically, the naturally occurring osteogenic proteinsare translated as a precursor, having an N-terminal signal peptidesequence typically less than about 30 residues, followed by a “pro”domain that is cleaved to yield the mature C-terminal domain. The signalpeptide is cleaved rapidly upon translation, at a cleavage site that canbe predicted in a given sequence using the method of Von Heijne (1986)Nucleic Acids Research 14:4683-4691. The pro domain typically is aboutthree times larger than the fully processed mature C-terminal domain.

[0074] Morphogens comprise a pair of polypeptide chains that, whenfolded, adopt a configuration sufficient for the resulting dimericprotein to elicit morphogenetic responses in cells and tissue displayingreceptors specific for said morphogen. That is, morphogens generallyinduce all of the following biological functions in a morphogenicallypermissive environment: stimulating proliferation of progenitor cells;stimulating the differentiation of progenitor cells; stimulating theproliferation of differentiated cells; and supporting the growth andmaintenance of differentiated cells. “Progenitor” cells are uncommittedcells that are competent to differentiate into one or more specifictypes of differentiated cells, depending on their genomic repertoire andthe tissue specificity of the permissive environment in whichmorphogenesis is induced. Morphogens further can delay or mitigate theonset of senescence- or quiescence-associated loss of phenotype and/ortissue function. Morphogens still further can stimulate phenotypicexpression of differentiated cells, including expression of metabolicand/or functional, e.g., secretory, properties thereof. In addition,morphogens can induce redifferentiation of committed cells underappropriate environmental conditions. As noted above, a morphogen thatinduces proliferation and/or differentiation of at least of dentin,cardiac, lung, liver, renal, adrenal, thyroid, ovarian, spleen, neural,pancreas, or gastrointestinal tract tissue, and/or support the growth,maintenance and/or functional properties of any of these tissues, is ofparticular interest herein.

[0075] Morphogenic proteins useful herein include any knownnaturally-occurring native proteins including allelic, phylogeneticcounterpart and other variants thereof, whether naturally-occurring orbiosynthetically produced (e.g., including “muteins” or “mutantproteins”), as well as new, biologically active members of the generalmorphogenic family of proteins.

[0076] Particularly useful sequences include those comprising theC-terminal 96 or 102 amino acid sequences of DPP (from Drosophila), Vg1(from Xenopus), Vgr-1 (from mouse), the OP1 and OP2 proteins, proteins(see U.S. Pat. No. 5,011,691 and Oppermann et al., as well as theproteins referred to as BMP2, BMP3, BMP4 (see WO88/00205, U.S. Pat. No.5,013,649 and W091/18098), BMP5 and BMP6 (see WO90/11366,PCT/US90/01630), BMP8 and BMP9. Other proteins useful in the practice ofthe invention include active forms of OP1, OP2, OP3, BMP2, BMP3, BMP4,BMP5, BMP6, BMP9, GDF-5, GDF-6, GDF-7, DPP, Vg1, Vgr, 60A protein,GDF-1, GDF-3, GDF-5, GDF-6, GDF-7, BMP10, BMP11, BMP13, BMP15, UNIVIN,NODAL, SCREW, ADMP or NURAL and amino acid sequence variants thereof. Inone currently preferred embodiment, morphogenic protein include any oneof: OP1, OP2, OP3, BMP2, BMP4, BMP5, BMP6, BMP9, and amino acid sequencevariants and homologs thereof, including species homologs, thereof.

[0077] Publications disclosing these sequences, as well as theirchemical and physical properties, include: OP-1 and OP-2: U.S. Pat. No.5,011,691, U.S. Pat. No. 5,266,683, Ozkaynak et al. (1990) EMBO J9:2085-2093; OP-3: WO94/10203 (PCT US93/10520); BMP2, BMP3, BMP4:WO88/00205, Wozney et al. (1988) Science 242:1528-1534); BMP5 and BMP6:Celeste et al. (1991) PNAS 87:9843-9847; Vgr-1: Lyons et al. (1989) PNAS86: 4554-4558; DPP: Padgett et al. (1987) Nature 325:81-84; Vg-1: Weeks(1987) Cell 51:861-867; BMP-9: WO95/33830 (PCT/US95/07084); BMP10:WO94/26893 (PCT/US94/05290); BMP-11: WO94/26892 (PCT/US94/05288); BMP12:WO95/16035 (PCT/US94/14030); BMP-13: WO95/16035 (PCT/US94/14030); GDF-1:WO92/00382 (PCT/US91/04096) and Lee et al. (1991) PNAS 88:4250-4254;GDF-8: WO94/21681 (PCT/US94/03019); GDF-9: WO94/15966 (PCT/US94/00685);GDF-10: WO95/10539 (PCT/US94/11440); GDF-11: WO96/01845(PCT/US95/08543); BMP-15: WO96/36710 (PCT/US96/06540); MP121: WO96/01316(PCT/EP95/02552); GDF-5 (CDMP-1, MP52): WO94/15949 (PCT/US94/00657) andWO96/14335 (PCT/US94/12814) and WO93/16099 (PCT/EP93/00350) and Storm etal., (1994), Nature 368:639-643; GDF-6 (CDMP-2, BMP13): WO95/01801(PCT/US94/07762) and WO96/14335 and WO95/10635 (PCT/US94/14030); GDF-7(CDMP-3, BMP12): WO95/10802 (PCT/US94/07799) and WO95/10635(PCT/US94/14030); BMP-3b: Takao, et al., (1996), Biochem. Biophys. Res.Comm. 219:656-662; GDF-3: WO94/15965; 60A: Blaster et al., (1993), Cell73:687-702 and GenBank accession number L12032. In another embodiment,useful proteins include biologically active biosynthetic constructs,including novel biosynthetic proteins and chimeric proteins designedusing sequences from two or more known osteogenic proteins. See also thebiosynthetic constructs disclosed in U.S. Pat. No. 5,011,691, thedisclosure of which is incorporated herein by reference (e.g., COP-1,COP-3, COP-4, COP-5, COP-7, and COP-16).

[0078] In certain preferred embodiments, morphogenic proteins usefulherein include those in which the amino acid sequences comprise asequence sharing at least 70% amino acid sequence homology or“similarity”, and preferably 80% homology or similarity, with areference morphogenic protein selected from the foregoing naturallyoccurring proteins. Preferably, the reference protein is human OP-1, andthe reference sequence thereof is the C-terminal seven cysteine domainpresent in morphogenically active forms of human OP-1, residues 330-431of SEQ ID NO:2. It will be appreciated that other known morphogenicproteins also can be used as the reference sequence. In one embodiment,a candidate amino acid sequence thought to be functionally equivalent toa reference amino acid sequence can be aligned therewith using themethod of Needleman, et al. (1970) J. Mol. Biol. 48:443-453, implementedconveniently by computer programs such as the Align program (DNAstar,Inc.). Internal gaps and amino acid insertions in the candidate sequenceare ignored for purposes of calculating the defined relationship,conventionally expressed as a level of amino acid sequence homology oridentity, between the candidate and reference sequences.

[0079] “Amino acid sequence homology” is understood herein to includeboth amino acid sequence identity and similarity. Homologous sequencesshare identical and/or similar amino acid residues, where similarresidues are conservative substitutions for, or “allowed pointmutations” of, corresponding amino acid residues in an aligned referencesequence. Thus, a candidate polypeptide sequence that shares 70% aminoacid homology with a reference sequence is one in which any 70% of thealigned residues are either identical to, or are conservativesubstitutions of, the corresponding residues in a reference sequence.Certain particularly preferred morphogenic polypeptide chains share atleast 60% amino acid sequence identity with the C terminal sevencysteine domain of the preferred reference sequence of human OP-1, stillmore preferably at least 65% amino acid sequence identity therewith.

[0080] Significant amino acid changes can be made from the referencesequence while retaining morphogenic activity. For example, while theGDF-1 protein sequence shares only about 50% amino acid identity withthe hOP-1 sequence described herein, the GDF-1 sequence shares greaterthan 70% amino acid sequence homology with the hOP-1 sequence, where“homology” is as defined above. Moreover, GDF-1 contains a four aminoacid insert (Gly-Gly-Pro-Pro) between the two residues corresponding toresidue 372 and 373 of OP-1 (SEQ ID NO:2). Similarly, BMP-3 has a“extra” residue, a valine, inserted between the two residuescorresponding to residues 385 and 386 of hOP-1 (SEQ ID NO:2). Also,BMP-2 and BMP-4 both are “missing” the amino acid residue correspondingto residue 389 of OP-1 (SEQ ID NO:2). None of these “deviations” fromthe reference sequence appear to interfere with biological activity.

[0081] As will be understood by those skilled in the art, homologous orfunctionally equivalent sequences include functionally equivalentarrangements of the cysteine residues within the conserved cysteineskeleton, including amino acid insertions or deletions which alter thelinear arrangement of these cysteines, but do not materially impairtheir relationship in the folded structure of the dimeric protein,including their ability to form such intra- or inter-chain disulfidebonds as may be necessary for biological activity. For example,naturally occurring morphogens have been described in which at least oneinternal deletion (of one residue; BMP2) or insertion (of four residues;GDF-1) is present but does not abrogate biological activity.Functionally equivalent sequences further include those wherein one ormore amino acid residues differ from the corresponding residue of areference sequence, e.g., the C-terminal seven cysteine domain (alsoreferred to herein as the conserved seven cysteine skeleton) of humanOP-1, provided that this difference does not destroy tissue morphogenicactivity.

[0082] As used herein, “conservative substitutions” are residues thatare physically or functionally similar to the corresponding referenceresidues, e.g., that have similar size, shape, electric charge, chemicalproperties including the ability to form covalent or hydrogen bonds, orthe like. Particularly preferred conservative substitutions are thosefulfilling the criteria defined for an accepted point mutation inDayhoff et al. (1978), 5 Atlas of Protein Sequence and Structure, Suppl.3, ch. 22 (pp. 354-352), Natl. Biomed. Res. Found., Washington, D.C.20007. Examples of conservative substitutions include the substitutionof one amino acid for another with similar characteristics, e.g.,substitutions within the following groups are well-known: (a) valine,glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine; (d)aspartic acid, glutamic acid; (e) asparagine, glutamine; (f) serine,threonine; (g) lysine, arginine, methionine; and (h) phenylalanine,tyrosine. The term “conservative variant” or “conservative variation”also includes the use of a substituted amino acid in place of anunsubstituted parent amino acid in a given polypeptide chain, providedthat antibodies having binding specificity for the resulting substitutedpolypeptide chain also have binding specificity (i.e., “crossreact” or“immunoreact” with) the unsubstituted or parent polypeptide chain.

[0083] In other preferred embodiments, the family of morphogenicproteins useful in the present invention, and members thereof, aredefined by a generic amino acid sequence. For example, Generic Sequence7 (SEQ ID NO:4) and Generic Sequence 8 (SEQ ID NO:5) disclosed below,accommodate the homologies shared among preferred protein family membersidentified to date, including at least OP-1, OP-2, OP-3, CBMP-2A,CBMP-2B, BMP-3, 60A, DPP, Vg1, BMP-5, BMP-6, Vgr-1, and GDF-1. The aminoacid sequences for these proteins are described herein and/or in theart, as summarized above. The generic sequences include both the aminoacid identity shared by these sequences in the C-terminal domain,defined by the six and seven cysteine skeletons (Generic Sequences 7 and8, respectively), as well as alternative residues for the variablepositions within the sequence. The generic sequences provide anappropriate cysteine skeleton where inter- or intrarnolecular disulfidebonds can form, and contain certain specified amino acids that mayinfluence the tertiary structure of the folded proteins. In addition,the generic sequences allow for an additional cysteine at position 36(Generic Sequence 7) or position 41 (Generic Sequence 8), therebyencompassing the biologically active sequences of OP-2 and OP-3. GenericSequence 7     Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa Xaa Xaa XaaXaa Xaa      1               5                   10                 15Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Pro Xaa Xaa             20                 25                   30 Xaa Xaa Xaa XaaXaa Xaa Asn His Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa35                   40                  45                  50 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa         55                  60                  65                  70Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa Xaa                 75                  80                  85 Met Xaa ValXaa Xaa Cys Xaa Cys Xaa      90                  95

[0084] wherein each Xaa independently is selected from a group of one ormore specified amino acids defined as follows: “Res.” means “residue”and Xaa at res.2=(Tyr or Lys); Xaa at res.3=Val or Ile); Xaa atres.4=(Ser, Asp or Glu); Xaa at res.6=(Arg, Gln, Ser, Lys or Ala); Xaaat res.7=(Asp or Glu); Xaa at res.8=(Leu, Val or Ile); Xaa atres.11=(Gln, Leu, Asp, His, Asn or Ser); Xaa at res. 12=(Asp, Arg, Asnor Glu); Xaa at res. 13=(Trp or Ser); Xaa at res.14=(Ile or Val); Xaa atres. 15=(Ile or Val); Xaa at res. 16=(Ala or Ser); Xaa at res. 18=(Glu,Gln, Leu, Lys, Pro or Arg); Xaa at res.19=(Gly or Ser); Xaa atres.20=(Tyr or Phe); Xaa at res.21=(Ala, Ser, Asp, Met, His, Gln, Leu orGly); Xaa at res.23=(Tyr, Asn or Phe); Xaa at res.26=(Glu, His, Tyr,Asp, Gln, Ala or Ser); Xaa at res.28=(Glu, Lys, Asp, Gln or Ala); Xaa atres.30=(Ala, Ser, Pro, Gln, Ile or Asn); Xaa at res.31=(Phe, Leu orTyr); Xaa at res.33=(Leu, Val or Met); Xaa at res.34=(Asn, Asp, Ala, Thror Pro); Xaa at res.35=(Ser, Asp, Glu, Leu, Ala or Lys); Xaa atres.36=(Tyr, Cys, His, Ser or Ile); Xaa at res.37=(Met, Phe, Gly orLeu); Xaa at res.38=(Asn, Ser or Lys); Xaa at res.39=(Ala, Ser, Gly orPro); Xaa at res.40=(Thr, Leu or Ser); Xaa at res.44=(Ile, Val or Thr);Xaa at res.45=(Val, Leu, Met or Ile); Xaa at res.46=(Gln or Arg); Xaa atres.47=(Thr, Ala or Ser); Xaa at res.48=(Leu or Ile); Xaa at res.49=(Valor Met); Xaa at res.50=(His, Asn or Arg); Xaa at res.51=(Phe, Leu, Asn,Ser, Ala or Val); Xaa at res.52=(Ile, Met, Asn, Ala, Val, Gly or Leu);Xaa at res.53=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.54=(Pro, Seror Val); Xaa at res.55=(Glu, Asp, Asn, Gly, Val, Pro or Lys); Xaa atres.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Gly, Ile or His); Xaa atres.57=(Val, Ala or Ile); Xaa at res.58=(Pro or Asp); Xaa atres.59=(Lys, Leu or Glu); Xaa at res.60=(Pro, Val or Ala); Xaa atres.63=(Ala or Val); Xaa at res.65=(Thr, Ala or Glu); Xaa atres.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa atres.68=(Asn, Ser, Asp or Gly); Xaa at res.69=(Ala, Pro or Ser); Xaa atres.70=(Ile, Thr, Val or Leu); Xaa at res.71=(Ser, Ala or Pro); Xaa atres.72=(Val, Leu, Met or Ile); Xaa at res.74=(Tyr or Phe); Xaa atres.75=(Phe, Tyr, Leu or His); Xaa at res.76=(Asp, Asn or Leu); Xaa atres.77=(Asp, Glu, Asn, Arg or Ser); Xaa at res.78=(Ser, Gln, Asn, Tyr orAsp); Xaa at res.79=(Ser, Asn, Asp, Glu or Lys); Xaa at res.80=(Asn, Thror Lys); Xaa at res.82=(Ile, Val or Asn); Xaa at res.84=(Lys or Arg);Xaa at res.85=(Lys, Asn, Gln, His, Arg or Val); Xaa at res.86=(Tyr, Gluor His); Xaa at res.87=(Arg, Gln, Glu or Pro); Xaa at res.88=(Asn, Glu,Trp or Asp); Xaa at res.90=(Val, Thr, Ala or Ile); Xaa at res.92=(Arg,Lys, Val, Asp, Gln or Glu); Xaa at res.93=(Ala, Gly, Glu or Ser); Xaa atres.95=(Gly or Ala) and Xaa at res.97=(His or Arg).

[0085] Generic Sequence 8 (SEQ ID NO:5) includes all of Generic Sequence7 and in addition includes the following five amino acid sequences (SEQID NO:8) at its N-terminus: Cys Xaa Xaa Xaa Xaa   1               5

[0086] Accordingly, beginning with residue 7, each “Xaa” in GenericSequence 8 is a specified amino acid defined as for Generic Sequence 7,with the distinction that each residue number described for GenericSequence 7 is shifted by five in Generic Sequence 8. Thus, “Xaa atres.2=(Tyr or Lys)” in Generic Sequence 7 refers to Xaa at res. 7 inGeneric Sequence 8. In Generic Sequence 8, Xaa at res.2=(Lys, Arg, Alaor Gln); Xaa at res.3=(Lys, Arg or Met); Xaa at res.4=(His, Arg or Gln);and Xaa at res.5=(Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr).

[0087] In another embodiment, useful morphogenic proteins include thosedefined by Generic Sequences 9 and 10, defined as follows.

[0088] Specifically, Generic Sequences 9 and 10 are composite amino acidsequences of the following proteins: human OP-1, human OP-2, human OP-3,human BMP-2, human BMP-3, human BMP-4, human BMP-5, human BMP-6, humanBMP-8, human BMP-9, human BMP10, human BMP-11, Drosophila 60A, XenopusVg-1, sea urchin UNIVIN, human CDMP-1 (mouse GDF-5), human CDMP-2 (mouseGDF-6, human BMP-13), human CDMP-3 (mouse GDF-7, human BMP-12), mouseGDF-3, human GDF-1, mouse GDF-1, chicken DORSALIN, dpp, DrosophilaSCREW, mouse NODAL, mouse GDF-8, human GDF-8, mouse GDF-9, mouse GDF-10,human GDF-11, mouse GDF-11, human BMP-15, and rat BMP3b. Like GenericSequence 7, Generic Sequence 9 accommodates the C-terminal six cysteineskeleton and, like Generic Sequence 8, Generic Sequence 10 accommodatesthe seven cysteine skeleton. Generic Sequence 9     Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa (SEQ ID NO:6)      1               5                  10                  15 Pro XaaXaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Gly Xaa Cys Xaa Xaa Xaa Xaa             20                  25                  30 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa     35                  40                  45                  50 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa                 55                  60                  65 Xaa Xaa XaaXaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa         70                  75                  80 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Cys Xaa Cys Xaa 85                  90                  95

[0089] wherein each Xaa is independently selected from a group of one ormore specified amino acids defined as follows: “Res.” means “residue”and Xaa at res. 1=(Phe, Leu or Glu); Xaa at res. 2=(Tyr, Phe, His, Arg,Thr, Lys, Gln, Val or Glu); Xaa at res. 3=(Val, Ile, Leu or Asp); Xaa atres. 4=(Ser, Asp, Glu, Asn or Phe); Xaa at res. 5=(Phe or Glu); Xaa atres. 6=(Arg, Gln, Lys, Ser, Glu, Ala or Asn); Xaa at res. 7=(Asp, Glu,Leu, Ala or Gln); Xaa at res. 8=(Leu, Val, Met, Ile or Phe); Xaa at res.9=(Gly, His or Lys); Xaa at res. 10=(Trp or Met); Xaa at res. 11=(Gln,Leu, His, Glu, Asn, Asp, Ser or Gly); Xaa at res. 12=(Asp, Asn, Ser,Lys, Arg, Glu or His); Xaa at res. 13=(Trp or Ser); Xaa at res. 14=(Ileor Val); Xaa at res. 15=(Ile or Val); Xaa at res. 16=(Ala, Ser, Tyr orTrp); Xaa at res. 18=(Glu, Lys, Gln, Met, Pro, Leu, Arg, His or Lys);Xaa at res. 19=(Gly, Glu, Asp, Lys, Ser, Gln, Arg or Phe); Xaa at res.20=(Tyr or Phe); Xaa at res. 21=(Ala, Ser, Gly, Met, Gln, His, Glu, Asp,Leu, Asn, Lys or Thr); Xaa at res. 22=(Ala or Pro); Xaa at res. 23=(Tyr,Phe, Asn, Ala or Arg); Xaa at res. 24=(Tyr, His, Glu, Phe or Arg); Xaaat res. 26=(Glu, Asp, Ala, Ser, Tyr, His, Lys, Arg, Gln or Gly); Xaa atres. 28=(Glu, Asp, Leu, Val, Lys, Gly, Thr, Ala or Gln); Xaa at res.30=(Ala, Ser, Ile, Asn, Pro, Glu, Asp, Phe, Gln or Leu); Xaa at res.31=(Phe, Tyr, Leu, Asn, Gly or Arg); Xaa at res. 32=(Pro, Ser, Ala orVal); Xaa at res. 33=(Leu, Met, Glu, Phe or Val); Xaa at res. 34=(Asn,Asp, Thr, Gly, Ala, Arg, Leu or Pro); Xaa at res. 35=(Ser, Ala, Glu,Asp, Thr, Leu, Lys, Gln or His); Xaa at res. 36=(Tyr, His, Cys, Ile,Arg, Asp, Asn, Lys, Ser, Glu or Gly); Xaa at res. 37=(Met, Leu, Phe,Val, Gly or Tyr); Xaa at res. 38=(Asn, Glu, Thr, Pro, Lys, His, Gly,Met, Val or Arg); Xaa at res. 39=(Ala, Ser, Gly, Pro or Phe); Xaa atres. 40=(Thr, Ser, Leu, Pro, His or Met); Xaa at res. 41=(Asn, Lys, Val,Thr or Gln); Xaa at res. 42=(His, Tyr or Lys); Xaa at res. 43=(Ala, Thr,Leu or Tyr); Xaa at res. 44=(Ile, Thr, Val, Phe, Tyr, Met or Pro); Xaaat res. 45=(Val, Leu, Met, Ile or His); Xaa at res. 46=(Gln, Arg orThr); Xaa at res. 47=(Thr, Ser, Ala, Asn or His); Xaa at res. 48=(Leu,Asn or Ile); Xaa at res. 49=(Val, Met, Leu, Pro or Ile); Xaa at res.50=(His, Asn, Arg, Lys, Tyr or Gln); Xaa at res. 51=(Phe, Leu, Ser, Asn,Met, Ala, Arg, Glu, Gly or Gln); Xaa at res. 52=(Ile, Met, Leu, Val,Lys, Gln, Ala or Tyr); Xaa at res. 53=(Asn, Phe, Lys, Glu, Asp, Ala,Gln, Gly, Leu or Val); Xaa at res. 54=(Pro, Asn, Ser, Val or Asp); Xaaat res. 55=(Glu, Asp, Asn, Lys, Arg, Ser, Gly, Thr, Gln, Pro or His);Xaa at res. 56=(Thr, His, Tyr, Ala, Ile, Lys, Asp, Ser, Gly or Arg); Xaaat res. 57=(Val, Ile, Thr, Ala, Leu or Ser); Xaa at res. 58=(Pro, Gly,Ser, Asp or Ala); Xaa at res. 59=(Lys, Leu, Pro, Ala, Ser, Glu, Arg orGly); Xaa at res. 60=(Pro, Ala, Val, Thr or Ser); Xaa at res. 61=(Cys,Val or Ser); Xaa at res. 63=(Ala, Val or Thr); Xaa at res. 65=(Thr, Ala,Glu, Val, Gly, Asp or Tyr); Xaa at res. 66=(Gln, Lys, Glu, Arg or Val);Xaa at res. 67=(Leu, Met, Thr or Tyr); Xaa at res. 68=(Asn, Ser, Gly,Thr, Asp, Glu, Lys or Val); Xaa at res. 69=(Ala, Pro, Gly or Ser); Xaaat res. 70=(Ile, Thr, Leu or Val); Xaa at res. 71=(Ser, Pro, Ala, Thr,Asn or Gly); Xaa at res. 2=(Val, Ile, Leu or Met); Xaa at res. 74=(Tyr,Phe, Arg, Thr, Tyr or Met); Xaa at res. 75=(Phe, Tyr, His, Leu, Ile,Lys, Gln or Val); Xaa at res. 76=(Asp, Leu, Asn or Glu); Xaa at res.77=(Asp, Ser, Arg, Asn, Glu, Ala, Lys, Gly or Pro); Xaa at res. 78=(Ser,Asn, Asp, Tyr, Ala, Gly, Gln, Met, Glu, Asn or Lys); Xaa at res.79=(Ser, Asn, Glu, Asp, Val, Lys, Gly, Gln or Arg); Xaa at res. 80=(Asn,Lys, Thr, Pro, Val, Ile, Arg, Ser or Gln); Xaa at res. 81=(Val, Ile, Thror Ala); Xaa at res. 82=(Ile, Asn, Val, Leu, Tyr, Asp or Ala); Xaa atres. 83=(Leu, Tyr, Lys or Ile); Xaa at res. 84=(Lys, Arg, Asn, Tyr, Phe,Thr, Glu or Gly); Xaa at res. 85=(Lys, Arg, His, Gln, Asn, Glu or Val);Xaa at res. 86=(Tyr, His, Glu or Ile); Xaa at res. 87=(Arg, Glu, Gln,Pro or Lys); Xaa at res. 88=(Asn, Asp, Ala, Glu, Gly or Lys); Xaa atres. 89=(Met or Ala); Xaa at res. 90=(Val, Ile, Ala, Thr, Ser or Lys);Xaa at res 91=(Val or Ala); Xaa at res. 92=(Arg, Lys, Gln, Asp, Glu,Val, Ala, Ser or Thr); Xaa at res. 93=(Ala, Ser, Glu, Gly, Arg or Thr);Xaa at res. 95=(Gly, Ala or Thr); Xaa at res. 97=(His, Arg, Gly, Leu orSer). Further, after res. 53 in rBMP3b and mGDF-10 there is an Ile;after res. 54 in GDF-1 there is a T; after res. 54 in BMP3 there is a V;after res. 78 in BMP-8 and Dorsalin there is a G; after res. 37 inhGDF-1 there is Pro, Gly, Gly, Pro.

[0090] Generic Sequence 10 (SEQ ID NO:7) includes all of GenericSequence 9 (SEQ ID NO:6) and in addition includes the following sequence(SEQ ID NO: 9) at its N-terminus: Cys Xaa Xaa Xaa Xaa SEQ ID NO:9  1               5

[0091] Accordingly, beginning with residue 6, each “Xaa” in GenericSequence 10 is a specified amino acid defined as for Generic Sequence 9,with the distinction that each residue number described for GenericSequence 9 is shifted by five in Generic Sequence 10. Thus, “Xaa at res.1=(Tyr, Phe, His, Arg, Thr, Lys, Gln, Val or Glu)” in Generic Sequence 9refers to Xaa at res. 6 in Generic Sequence 10. In Generic Sequence 10,Xaa at res. 2=(Lys, Arg, Gln, Ser, His, Glu, Ala, or Cys); Xaa at res.3=(Lys, Arg, Met, Lys, Thr, Leu, Tyr, or Ala); Xaa at res. 4=(His, Gln,Arg, Lys, Thr, Leu, Val, Pro, or Tyr); and Xaa at res. 5=(Gln, Thr, His,Arg, Pro, Ser, Ala, Gln, Asn, Tyr, Lys, Asp, or Leu).

[0092] As noted above, certain currently preferred bone morphogenicpolypeptide sequences useful in this invention have greater than 60%identity, preferably greater than 65% identity, with the amino acidsequence defining the preferred reference sequence of hOP-1. Theseparticularly preferred sequences include allelic and phylogeneticcounterpart variants of the OP-1 and OP-2 proteins, including theDrosophila 60A protein. Accordingly, in certain particularly preferredembodiments, useful proteins include active proteins comprising pairs ofpolypeptide chains within the generic amino acid sequence hereinreferred to as “OPX” (SEQ ID NO:3), which defines the seven cysteineskeleton and accommodates the homologies between several identifiedvariants of OP-1 and OP-2. As described herein, each Xaa at a givenposition independently is selected from the residues occurring at thecorresponding position in the C-terminal sequence of mouse or human OP-1or OP-2.     Cys Xaa Xaa His Glu Leu Tyr Val Ser Phe Xaa Asp Leu Gly TrpXaa Asp Trp     1               5                  10                  15 Xaa IleAla Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly Glu Cys Xaa Phe Pro    20                   25                  30                  35 LeuXaa Ser Xaa Met Asn Ala Thr Asn His Ala Ile Xaa Gln Xaa Leu Val His Xaa        40                  45                   50                  55Xaa Xaa Pro Xaa Xaa Val Pro Lys Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala            60                   65                  70 Xaa Ser Val LeuTyr Xaa Asp Xaa Ser Xaa Asn Val Ile Leu Xaa Lys Xaa Arg75                   80                  85                  90 Asn MetVal Val Xaa Ala Cys Gly Cys His          95                 100

[0093] wherein Xaa at res. 2=(Lys or Arg); Xaa at res. 3=(Lys or Arg);Xaa at res. 11=(Arg or Gln); Xaa at res. 16=(Gln or Leu); Xaa at res.19=(Ile or Val); Xaa at res. 23=(Glu or Gln); Xaa at res. 26=(Ala orSer); Xaa at res. 35=(Ala or Ser); Xaa at res. 39=(Asn or Asp); Xaa atres. 41=(Tyr or Cys); Xaa at res. 50=(Val or Leu); Xaa at res. 52=(Seror Thr); Xaa at res. 56=(Phe or Leu); Xaa at res. 57=(Ile or Met); Xaaat res. 58=(Asn or Lys); Xaa at res. 60=(Glu, Asp or Asn); Xaa at res.61=(Thr, Ala or Val); Xaa at res. 65=(Pro or Ala); Xaa at res. 71=(Glnor Lys); Xaa at res. 73=(Asn or Ser); Xaa at res. 75=(Ile or Thr); Xaaat res. 80=(Phe or Tyr); Xaa at res. 82=(Asp or Ser); Xaa at res.84=(Ser or Asn); Xaa at res. 89=(Lys or Arg); Xaa at res. 91=(Tyr orHis); and Xaa at res. 97=(Arg or Lys).

[0094] In still another preferred embodiment, useful morphogenicallyactive proteins have polypeptide chains with amino acid sequencescomprising a sequence encoded by a nucleic acid that hybridizes, underlow, medium or high stringency hybridization conditions, to DNA or RNAencoding reference morphogenic sequences, e.g., C-terminal sequencesdefining the conserved seven cysteine domains of OP-1, OP-2, BMP2, BMP4,BMP5, BMP6, 60A, GDF5, GDF6, GDF7 and the like. As used herein, highstringent hybridization conditions are defined as hybridizationaccording to known techniques in 40% formamide, 5× SSPE, 5× Denhardt'sSolution, and 0.1% SDS at 37° C. overnight, and washing in 0.1× SSPE,0.1% SDS at 50° C. Standard stringency conditions are well characterizedin commercially available, standard molecular cloning texts. See, forexample, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. bySambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press:1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985);Oligonucleotide Synthesis (M. J. Gait ed., 1984): Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); and B. Perbal, APractical Guide To Molecular Cloning (1984).

[0095] The morphogenic proteins contemplated herein can be expressedfrom intact or truncated genomic or cDNA or from synthetic DNAs inprokaryotic or eukaryotic host cells. The dimeric proteins can beisolated from the culture media and/or refolded and dimerized in vitroto form biologically active compositions. Heterodimers can be formed invitro by combining separate, distinct polypeptide chains. Alternatively,heterodimers can be formed in a single cell by co-expressing nucleicacids encoding separate, distinct polypeptide chains. See, for example,WO93/09229, or U.S. Pat. No. 5,411,941, for several exemplaryrecombinant heterodimer protein production protocols. Currentlypreferred host cells include, without limitation, prokaryotes includingE. coli, or eukaryotes including yeast or Saccharomvces, or mammaliancells, such as CHO, COS or BSC cells. One of ordinary skill in the artwill appreciate that other host cells can be used to advantage. Detaileddescriptions of the proteins useful in the practice of this invention,including how to make, use and test them for morphogenic activity, aredisclosed in numerous publications, including U.S. Pat. Nos. 5,266,683,5,011,691, and/or U.S. Pat. No. 5,585,237, the disclosures of which areincorporated by reference herein, as well as in any of the publicationsrecited herein, including any of the U.S. patents, the disclosures ofwhich are incorporated herein by reference.

[0096] The dimeric protein species described herein above are referredto herein as “mature” morphogenic proteins. Soluble forms of theseproteins also can be created by complexing the dimeric species with partor all of at least one, and preferably two morphogenic protein prodomain peptides. Alternatively, a soluble complex form of a morphogenicprotein can be isolated from the cell culture media using the protocoldescribed in WO94/03600, published Feb. 18, 1994, for example. (Seebelow).

[0097] Other soluble forms of morphogens include dimers of the uncleavedpro forms of these proteins, as well as “hemi-dimers” wherein onesubunit of the dimer is an uncleaved pro form of the protein, and theother subunit comprises the mature form of the protein, includingtruncated forms thereof, preferably noncovalently associated with acleaved pro domain peptide.

[0098] As described in published application WO94/03600, the teachingsof which are incorporated herein by reference, useful pro domainsinclude the full length pro regions, as well as various truncated formshereof, particularly truncated forms cleaved at proteolyticArg-Xaa-Xaa-Arg cleavage sites within the pro domain polypeptide. Forexample, in OP1, possible pro sequences include sequences defined byresidues 30-292 (full length form); 48-292; and 158-292, all of SEQ IDNO:2. Soluble OP1 complex stability is best enhanced when the pro regioncomprises the full length form rather than a truncated form, such as theresidues 48-292 truncated form, in that residues 30-47 show sequencehomology to the N-terminal portions of other morphogens, and currentlyare believed to have particular utility in enhancing complex stabilityfor all morphogens. Accordingly, currently preferred pro domains includepeptides comprising at least the N-terminal fragment, e.g., amino acidresidues 30-47 of a naturally occurring morphogen pro domain, or abiosynthetic variant thereof that retains the solubility and/orstability enhancing properties of the naturally-occurring peptide.

[0099] As will be appreciated by those having ordinary skill in the art,useful sequences encoding the pro region can be obtained from geneticsequences encoding known morphogens. Alternatively, chimeric pro regionscan be constructed from the sequences of one or more known morphogens.Still another option is to create a synthetic sequence variant of one ormore known pro region sequences.

[0100] Soluble morphogen complexes can be isolated from conditionedmedia using a simple, three step chromatographic protocol performed inthe absence of denaturants. The protocol involves running the media (orbody fluid) over an affinity column, followed by ion exchange and gelfiltration chromatographies. The affinity column described below is aZn-IMAC column. An alternative protocol also envisioned to have utilityincludes an immunoaffinity column, created using standard proceduresand, for example, using antibody specific for a given morphogen prodomain (complexed, for example, to a protein A-conjugated Sepharosecolumn). Protocols for developing immunoaffinity columns are welldescribed in the art (see, for example, Guide to Protein Purification,M. Deutscher, ed., Academic Press, San Diego, 1990, particularlysections VII and XI thereof).

[0101] Morphogens can be expressed in any suitable host cell competentto express recombinant protein. For example, mammalian (CHO, Chinesehamster ovary) cells as described in the art (see, for example,international application US90/05903 (WO91/05802) can be used. The CHOcell conditioned media containing 0.5% FBS is initially purified usingImmobilized Metal-Ion Affinity Chromatography (IMAC). The solublemorphogen complex from conditioned media binds very selectively to theZn-IMAC resin and a high concentration of imidazole (50 mM imidazole, pH8.0) is required for the effective elution of the bound complex. TheZn-IMAC step separates the soluble morphogen from the bulk of thecontaminating serum proteins that elute in the flowthrough and 35 mMimidazole wash fractions. The Zn-IMAC purified soluble morphogen is nextapplied to an S-Sepharose cation-exchange column equilibrated in 20 mMNaPO₄ (pH 7.0) with 50 mM NaCl. This S-Sepharose step serves to furtherpurify and concentrate the soluble complex in preparation for thefollowing gel filtration step. The protein was applied to a SephacrylS-200HR column equilibrated in TBS. Using substantially the sameprotocol, soluble morphogens also can be isolated from one or more bodyfluids, including serum, cerebrospinal fluid or peritoneal fluid.

[0102] The soluble complex isolated from cell culture media elutes withan apparent molecular weight of 110 kDa as compared with proteinmolecular weight standards. The identity of the proteins can beconfirmed by N-terminal sequencing. Purity of the final complex can beverified by running the appropriate fraction in a reduced 15%polyacrylamide gel.

[0103] As an alternative to purifying soluble complexes from culturemedia or a body fluid, soluble complexes can be formulated from purifiedpro domains and mature dimeric species. Successful complex formationapparently requires association of the components under denaturingconditions sufficient to relax the folded structure of these molecules,without affecting disulfide bonds. Preferably, the denaturing conditionsmimic the environment of an intracellular vesicle sufficiently such thatthe cleaved pro domain has an opportunity to associate with the maturedimeric species under relaxed folding conditions. The concentration ofdenaturant in the solution then is decreased in a controlled, preferablystep-wise manner, so as to allow proper refolding of the dimer and proregions while maintaining the association of the pro domain with thedimer. Useful denaturants include 4-6M urea or guanidine hydrochloride(GuHCl), in buffered solutions of pH 4-10, preferably pH 6-8. Thesoluble complex then is formed by controlled dialysis or dilution into asolution having a final denaturant concentration of less than 0.1-2Murea or GuHCl, preferably 1-2 M urea of GuHCl, which then preferably canbe diluted into a physiological buffer. Protein purification/renaturingprocedures and considerations are well described in the art, and detailsfor developing a suitable renaturing protocol readily can be determinedby one having ordinary skill in the art. One useful text on the subjectis Guide to Protein Purification, M. Deutscher, ed., Academic Press, SanDiego, 1990, particularly section V. Complex formation also may be aidedby addition of one or more chaperone proteins. Stability of the complexalso is enhanced by presence of positively charged amino acids, such asarginine.

II. FORMULATION AND DELIVERY CONSIDERATIONS

[0104] General Considerations

[0105] The compositions useful in practice of the invention can beformulated using routine methods. All that is required is determinationof the desired final concentration of morphogenic protein peradministration. Useful formulation methodologies include solubilizationof lyophilized protein or analog. Useful protein or analogsolubilization solutions include ethanol, urea, physiological and/oracidic buffers, saline buffers, and acetonitrile/trifluoroacetic acidsolutions, and the like. See, for example, U.S. Pat. No. 5,266,683. Thedesired final concentration of protein or analog will depend on thespecific activity of the protein or analog as well as the type, volume,and/or anatomical location of the defect. In one preferred embodiment,useful proteins and analogs are those having a half maximal bone formingspecific activity of 1.0-2.0 ng molecule/25mg matrix, or 0.5-1.0 ngprotein/25 mg matrix as measured in a standard rat bioassay. Proteinshaving lower specific activity also can be used to advantage as canmorphogen having specific activity in a different tissue morphogensassay. Additionally, the desired final concentration of protein candepend on the age, sex and/or overall health of the recipient. All ofthese considerations can be evaluated and optimized using the assaysenabled by the invention.

[0106] Useful morphogen protein or analog dosage ranges are contemplatedto include 0.1-1000 mg/kg body weight, preferably in the range of 1-100mg/kg. As described herein below, protein or analog can be administeredsystemically as a single bolus or as multiple doses administered overtime. Useful concentrations for liquid administration include a rangefrom about 0.5-5000 ml. Optimization of dosages requires no more thanroutine experimentation and is within the skill level of one of ordinaryskill in the art. It should be noted that no obvious morphogenicprotein-induced pathological lesions arise when mature protein (e.g.,OP1, 20 mg) is administered daily to normal growing rats for 21consecutive days. Moreover, 10 mg systemic injections of morphogen(e.g., OP1) injected daily for 10 days into normal newborn mice does notproduce any gross abnormalities. Toxiaty levels of candidate analogsreadily can be determined by the instant invention to determinetherapeutically useful dosages.

[0107] The protein or analog can be provided to an individual by anymeans suitable for systemic administration, (e.g., parenterally, as byi.v. or intra peritoneally, or orally). Liquid formulations preferablycomprise part of an aqueous, physiologically acceptable solution so thatin addition to delivery of the desired protein or analog to a targetsite, the solution does not otherwise adversely affect the cells' orsubject's electrolyte and/or volume balance. Suitable aqueous mediumsinclude, without limitation, normal physiologic saline (e.g., 9.85%NaCl, 0.15M, pH 7-7.4). Such an aqueous solution containing the agentcan be made, for example, by dissolving lyophilized protein ordispersing the protein in 50% ethanol containing acetonitrile in 0.1%trifluoroacetic acid (TFA) or 0.1% HCl, or equivalent solvents. Onevolume of the resultant solution then is added, for example, to tenvolumes of phosphate buffered saline (PBS), which further may include0.1-0.2% human serum albumin (HSA). The resultant solution preferably isvortexed extensively. Alternatively, lyophilized protein or analog canbe solubilized in sodium acetate buffer (pH 4.5) or its equivalent.

[0108] Where the protein or analog is to be provided parenterally, suchas by intravenous, subcutaneous, intramuscular, intraorbital,ophthalmic, intraventricular, intracranial, intracapsular, intraspinal,intracisternal, intraperitoneal, buccal, rectal, vaginal, intranasal orby aerosol administration, the protein preferably comprises part of anaqueous solution. Currently preferred for intravenous administration ofa morphogen is PBS or a sodium acetate buffer. The protein or analog canbe administered as a single dose or by periodic injections of a bolus ofthe protein or analog, or can be made more continuous by intravenous orintraperitoneal administration from a reservoir which is external (e.g.,an i.v. bag) or internal (e.g., a bioerodable implant, or a colony ofimplanted, morphogen/analog-producing cells).

[0109] Useful solutions for parenteral administration may be prepared byany of the methods well known in the pharmaceutical art, described, forexample, in Remington's Pharmaceutical Sciences (Gennaro, A., ed.), MackPub., 1990.

[0110] Alternatively, the morphogenic protein or analog described hereincan be administered orally. Oral administration of proteins astherapeutics generally is not practiced as most proteins are readilydegraded by digestive enzymes and acids in the mammalian digestivesystem before they can be absorbed into the bloodstream. However, themorphogenic proteins described herein typically are acid stable andprotease-resistant (see, for example, U.S. Pat. No. 4,968,590). Inaddition, at least one morphogen, OP1, has been identified in mammarygland extract, colostrum and 57-day milk. Moreover, the OP1 purifiedfrom mammary gland extract is morphogenically active and also isdetected in the bloodstream. Maternal administration, via ingested milk,may be a natural delivery route of TGFβ superfamily proteins. Letterioet al. (1994), Science 264:1936-1938, report that TGFβ is present inmurine milk, and that radiolabeled TGFβ is absorbed by gastrointestinalmucosa of suckling juveniles. Labeled, ingested TGFβ appears rapidly inintact form in the juveniles' body tissues, including lung, heart andliver. These findings, as well as those disclosed in the examples below,indicate that oral and parenteral administration are viable means foradministering morphogenic proteins systemically to an individual. Inaddition, while the mature forms of certain morphogenic proteinsdescribed herein typically are sparingly soluble, the protein form foundin milk (and mammary gland extract and colostrum) is readily soluble,probably by association of the mature, dimeric species with part or allof the pro domain of the intact sequence and/or by association with oneor more milk components. See, for example, WO93/0575 1, published Apr.1, 1993 and U.S. Ser. No. 08/278,730, the disclosure of which isincorporated herein by reference. Accordingly, the compounds providedherein also can be associated with molecules capable of enhancing theirsolubility in vitro or in vivo.

[0111] Another molecule capable of enhancing solubility and particularlyuseful for oral administrations, is casein. For example, addition of0.2% casein increases solubility of the mature active form of OP1 by80%. Other components found in milk and/or various serum proteins alsomay be useful.

[0112] Alternatively or, in addition, orally administered morphogenicprotein or analog can be formulated as part of a delivery vehiclecompetent to be transported through the gastrointestinal system. As oneexample, the protein can be formulated as part of biologically erodiblemicrosphere, particularly one whose polymers have sufficient adhesiveproperties to allow temporary interaction with the gastrointestinalmucus and cellular linings and can traverse both the mucosal absorptiveepithelium and the follicle-associated epithelium covering the lymptoidtissue of Peyer's patches. Useful polymers include, without limitation,polystyrene, polylactide and/or polyglycolide and/or polycyanoacrylatepolymers and combinations thereof, and can be used to transportmorphogenic protein across the intestinal lining and into thecirculating system. The microspheres described below and in Mathlowitzet al. (1996) Nature 386: 410-414, also are considered to be useful.Here polyanhydride copolymers of fumaric and sebacic acid “poly(FA:SA)”,formulated into 20:80 mircospheres (diameter 0.1-10 mm) made, forexample by phase inversion nanoencapsulation, traverse both membranes inas little as one hour, as determined by optical microscopy. Still otheruseful microspheres include polymer blends of poly (fumaric anhydride)and poly (lactide-co-glycolide).

[0113] Morphogenic protein or analog readily can be microencapsulated bystandard phase inversion protocols. For example, morphogenic protein isadded to a dilute polymer solution (i.e., 1-4% w/v in methylenechloride), which then is poured rapidly into an unstirred bath ofnon-solvent (petroleum ether) at a solvent to non-solvent ratio of1:100, causing nano and microspheres (0.1-5.0 μm in diameter) to formspontaneously.

[0114] The morphogenic proteins and analogs, of course, can beadministered systemically alone or in combination with other moleculesknown to be beneficial in the treatment of the conditions describedherein. Thus, in other embodiments the present invention provides assaysfor evaluating pharmaceutical compositions in which an morphogenicprotein or analog is combined with other agents which promote or enhancenew tissue formation. In each such composition, the ratios of themorphogenic and mitogenic agents may be adjusted based upon theiractivities, as disclosed in the literature and as determined throughsimple experimentation using the methods of the instant invention, toprovide a therapeutically effective dosage of each compound in a singleunit dosage. The morphogenic and mitogenic agents in such a compositioneach preferably comprise at least about 1%, and more preferably morethan 5% or 10%, of the dry weight of the composition. The compositionscan, however, include other pharmaceutical carriers and active agents,as described above and, generally, in Remington's PharmaceuticalSciences (Gennaro, A, ed.), Mack Pub., 1990, and, therefore, themorphogenic and mitogenic agents can each comprise a small fraction ofthe final weight of the pharmaceutical composition.

[0115] Morphogenic formulations readily can be sterilized using standardprocedures prior to implantation. For example, proteins conveniently canbe filter-sterilized, e.g., using a 0.22 micron filter. Alternatively,chemicals, such as ethylene oxide can be used. Carrier materials,wetting agents and/or binding agents can be sterilized by exposure tochemicals, heat, or ionizing radiation. In addition, morphogenicformulations can be terminally sterilized to a sterility assurance levelof 10⁻⁶ by exposure to ionizing radiation, for example, gamma orelectron beam radiation. Useful dose ranges include within the range ofabout 0.5-4.0 megarads, preferably 2.0-3.5 megarads. See, for example,WO 96/40297, published Dec. 19, 1996.

[0116] Practice of the invention will be still more fully understoodfrom the following examples, which are presented herein for illustrationonly and should not be construed as limiting the invention in any way.

IV. BIOASSAY

[0117] A. Bioassay of Bone Morphogenic Activity: Endochondral BoneFormation and Related Properties

[0118] The art-recognized bioassay for bone induction as described bySampath and Reddi (Proc. Natl. Acad. Sci. USA (1983) 80:6591-6595) andU.S. Pat. No. 4,968,590, the disclosure of which is herein incorporatedby reference, can be used to establish the efficacy of a given device orformulation. Briefly, the assay consists of depositing test samples insubcutaneous sites in recipient rats under ether anesthesia. A verticalincision (1 cm) is made under sterile conditions in the skin over thethoracic region, and a pocket is prepared by blunt dissection. Incertain circumstances, approximately 25 mg of the test sample isimplanted deep into the pocket and the incision is closed with ametallic skin clip. The heterotropic site allows for the study of boneinduction without the possible ambiguities resulting from the use oforthotopic sites. The implants also can be provided intramuscularlywhich places the devices in closer contact with accessible progenitorcells. Typically intramuscular implants are made in the skeletal muscleof both legs.

[0119] The sequential cellular reactions occurring at the heterotropicsite are complex. The multistep cascade of endochondral bone formationincludes: binding of fibrin and fibronectin to implanted matrix,chemotaxis of cells, proliferation of fibroblasts, differentiation intochondroblasts, cartilage formation, vascular invasion, bone formation,remodeling, and bone marrow differentiation.

[0120] Successful implants exhibit a controlled progression through thestages of protein-induced endochondral bone development including: (1)transient infiltration by polymorphonuclear leukocytes on day one; (2)mesenchymal cell migration and proliferation on days two and three; (3)chondrocyte appearance on days five and six; (4) cartilage matrixformation on day seven; (5) cartilage calcification on day eight; (6)vascular invasion, appearance of osteoblasts, and formation of new boneon days nine and ten; (7) appearance of osteoblastic and bone remodelingon days twelve to eighteen; and (8) hematopoietic bone marrowdifferentiation in the ossicle on day twenty-one.

[0121] Histological sectioning and staining is preferred to determinethe extent of osteogenesis in the implants. Staining with toluidine blueor hemotoxylin/eosin clearly demonstrates the ultimate development ofendochondral bone. Twelve day bioassays are sufficient to determinewhether bone inducing activity is associated with the test sample.

[0122] Additionally, alkaline phosphatase activity and/or total calciumcontent can be used as biochemical markers for osteogenesis. Thealkaline phosphatase enzyme activity can be determinedspectrophotometrically after homogenization of the excised testmaterial. The activity peaks at 9-10 days in vivo and thereafter slowlydeclines. Samples showing no bone development by histology should haveno alkaline phosphatase activity under these assay conditions. The assayis useful for quantitation and obtaining an estimate of bone formationvery quickly after the test samples are removed from the rat. Theresults as measured by alkaline phosphatase activity level andhistological evaluation can be represented as “bone forming units”. Onebone forming unit represents the amount of protein that is needed forhalf maximal bone forming activity on day 12. Additionally, dose curvescan be constructed for bone inducing activity in vivo at each step of apurification scheme by assaying various concentrations of protein.Accordingly, the skilled artisan can construct representative dosecurves using only routine experimentation.

[0123] Total calcium content can be determined after homogenization in,for example, cold 0.15M NaCl, 3 mM NaHCO₃, pH 9.0, and measuring thecalcium content of the acid soluble fraction of sediment.

B. EXAMPLE 1

[0124] This assay demonstrates the ability of systemically administeredmorphogenic protein to induce bone formation at a local defect site. Alocal, permissive site was created by implanting matrix alone (bovinedemineralized, deproteinated bone collagen) at a subcontaneous orintramuscular site in a rat. Bone collagen, when implanted alone in asubcutaneous site, provokes localized inflammation, and rapidly becomessurrounded by migrating mesenchymal stem cells, e.g., progenitor cellsthat can respond to osteoinductive signals when given locally, to inducenew bone formation. Left alone, the induced inflammatory response leadsto fibrotic tissue formation at the implant site which can be resolvedover time. In the present example, buffer alone, soluble OP-1, or matureOP-1, were administered systemically to evaluate the ability ofmorphogenic protein to induce new bone formation at the implant locus.

[0125] Long-Evans rats each were subjected to four bone collagenimplants: Two subcutaneously (left, right sides of thoracic region) andtwo intramuscularly (one on muscle of each hind leg), as described above(25 mg matrix/implant). Soluble OP-1 (0.5 mg, mature equivalent in PBSbuffer) or mOP-1 (0.5 mg in acetate buffer) was administeredintravenously (i.v.) through tail vein at time 0, 8, 24, 48 and 72 hoursafter implantation. The animals were sacrificed on day 12 and examinedfor new bone formation by histology and biochemical markers. The day ofimplantation was considered as Day 0. Four groups of four rats each weretested as follows: Group Assay 1) none 2) buffer alone; five injections0, 8, 24, 48 and 72 hours (in 500 μl) 3) sOP-1 (500 μg); five injections@ 0, 8, 24, 48 and 72 hrs (in 500 μl) 4) mOP-1 (500 μg); five injections@ 0, 8, 24, 48 and 72 hrs (in 500 μl) Results. Units alk. phos./ μgcalcium/ Assay Histology* mg protein mg tissue negative control: none —<0.1 <5 buffer (NaAc) — 0.6 <5 positive control: matrix + +++ 0.8 >30sOP-1 (implant) matrix + mOP-1 1.5 >50 (implant) +++ systemic admin.:sOP-1 +/− 0.7 >30 mOP-1 ++ 2.1 >30

EXAMPLE 2

[0126] This example provides a protocol for evaluating the ability ofsystemically provided protein to target to the local defect sitefollowing systemic administration.

[0127] In this example, administered protein serum levels are evaluatedby standard protocols (Western blot, Elisa, and/or radio-iodination) attimes 0 min., 5 min. and 30 min. after injection.

[0128] Specifically, mOP-1 (2.5 mg mature in 500 μl of sodium acetate)is administered at 24h after collagen implant i.v. through tail vein.Appropriate controls include buffer alone. The animals are sacrificed at5 and 30 min. after OP-1 administration. The implants are extracted in 8M urea containing detergent buffer and the presence of OP-1 in theimplant is examined by Elisa and Western blot analyses. In a secondprotocol, iodinated OP-1 is provided and radioactive protein measured atthe implant site at 5′ and 30′ post administration.

[0129] In order to determine whether systemically-administered OP-1 isavailable in collagen implants, iodinated OP-1 was administered 24 hoursfollowing collagen implantation. The implants were then harvested 5, 15,and 60 minutes after administration of the labeled OP-1. Adjacentsubcutaneous fascia and thigh skeletal tissue were harvested ascontrols. SDS-PAGE autoradiography analysis of extracted tissue proteinsdemonstrated that a portion of the iodinated OP-1 was detectable in thecollagen implants harvested at 5 minutes, but was not detectable in theimplants harvested at 15 or 60 minutes. Control samples did not containdetectable levels of labeled OP-1 at any time interval. Previouspharmacokinetic studies have shown that about 0.5% ofintraveneously-administered OP-1 is available in circulation within oneminutes, and is cleared from circulation with a half-life of about 15-30minutes. It is likely that a portion of the OP-1 detectable at 5 minutesfollowing iv administration is sufficient to trigger differentiation ofendochondral bone.

[0130] Tests showed that bovine bone-derived insoluble type I collagenis superior to other insoluble collagens obtained from rat tail tendon,bovine achilles tendon and type IV collagen-enriched matrigel.Hydroxyapatite was also less effective as a carrier. Surgical woundsites without a collagen implant showed no signs of bone formation,indicating that collagen implants are required to recruit mesenchymalcells locally in order to respond to OP-1 signal upon systemicadministration.

EXAMPLE 3

[0131] This example demonstrates that a single dose of systemicallyadministered morphogenic protein is competent to induce new tissueformation at a locale permissive defect site distal to the site ofadministration, and that therapeutic effect is not time-sensitive.Specifically, morphogenic protein, administered as a single bolus attwenty-four or fourty-eight or seventy-two hrs. induces new boneformation in the rat bioassay, at levels comparable to those seen withmultiple injections.

[0132] In these studies OP-1 was administered systemically at a singleconcentration (500 μg in 500 μl, given 5 injections @ 0. 6, 24, 48 and72 hrs. after collagen matrix implantation, as shown in Example 1, orsingle administration of 500 μg of OP-1 at any given time). Controlgroups (none and buffer injected) did not induce bone formation at thecollagen implant sites (s.c. or i.m.) whereas mature OP-1 injectedgroups induced endochondral bone, as examined after 12 days.

[0133] Long-Evans (6 wks old) were subjected to subcutaneous andintramuscular implants as detailed above. The implants consisted ofbovine collagenous matrix (approximately 25 mg) only, implanted atsubcutaneous site on both side of the rats, and at skeletal muscle ofboth legs. Four implants were made per rat, with four rats per group.The day of collagen implant or OP-1 administration is considered as Day0 and Time 0 hrs.

[0134] This example demonstrates the ability of a single dose to affectnew bone formation in a mammal, and that the dose can be administered atlong times after the defect has been created. Specifically, systemicallyadministered morphogenic protein is effective even when provided afterthe onset or initiation of fibrotic tissue formation.

[0135] By seventy-two hours post trauma the microenvironment of a localdefect site has normalized and stabilized. Specifically, microvessalshave been repaired, any inflammatory response triggered by the surgeryor trauma has stabilized, and fibroblasts now are present at the siteand can initiate the laying down of extracellular matrix scarringtissue. Thus, in the absence of morphogenic protein, by seventy-two hrs.post implant the micro environment of a test implant now substantiallymimics that of a refractory healing site in a compromised individual,e.g., an individual whose ability to form new bone callus is compromisedby, for example, age, disease (for example, diabetes, osteoporosis),therapeutics used in association with surgical or other therapeuticprocedures (steroids, for example). The ability of systemicallyadministered morphogenic proteins to induce new bone formation whenadministered at times substantially after the defect has been createdhas positive implications for treating patients suffering fromrefractory healing.

[0136] Osteogenesis induced by OP-1 (2.5 mg/rat) was maximal when OP-1was administered 24 or 72 hours after collagen implantation. Anapproximately 30% reduction in osteogenesis was observed when OP-1 wasadministered 6 hours after implantation. FIG. 2 shows these results. Ineach case, OP-1 was administered at the time point indicated, andosteogenesis was measured at day 12 after OP-1 administration. At timeslonger than 120 hours post-implantation OP-1 failed to induce bone,suggesting that collagen may have already been committed to fibroblastlineages, and therefore non-responsive to OP-1.

EXAMPLE 4

[0137] This example demonstrates that bone formation is dependent on thedose of systemically administered morphogenic protein. Specifically, inthis assay mOP-1 (0.05, 0.5, 2.5 mg in 500 μl acetate buffer) andsoluble OP-1 (0.05, 0.5, 2.5 mg mature equivalent in PBS) wereadministered (at 24h after collagen implant) i.v. through tail vein.Appropriate controls include buffer (sodium acetate) alone. The animalswere sacrificed on day 12 after OP-1 administration and examined for newbone formation by histology, alkaline phosphatase activity and calciumcontent. Long-Evans (4-5 wks old) were subjected to subcutaneous andintramuscular implants as detailed above. Four implants per rat and fourrats per group. The day of OP-1 administration is considered as Day 0and Time 0 hrs.

[0138] Results. Control groups (buffer injected) did not induce boneformation at the collagen implant sites (s.c. or i.m.) whereas matureOP-1 injected groups induced endochondral bone, as examined after 12-14days. The bone forming activity exhibited by the group that receivedmultiple injections is comparable to that observed in Example 1. Theamount of bone forming activity observed in the groups that receivedsingle administration was directly related to the dosing of OP-1; withthe maximum activity observed in high concentration of OP-1 (2.5 mg),and exhibiting bone forming activity comparable to that observed inExample 1.

[0139] Groups that received a single administration of OP-1 at 24h aftercollagen implant, all exhibited bone formation, with the higher dosegroup (2.5 mg) showing bone formation comparable to the group thatreceived multiple injections; and the low concentration (50 or 500 ug)groups showing endochondral bone formation in 50 or 60% of the implantsrespectively. Since all implants from groups that received multiple or asingle high dose administration formed bone comparable to each other,the amount of bone formed by the systemic administration is notdependent on the dosing regiment. Based on the rat bioassay, a currentlypreferred dose range for effecting osteogenesis in a rat by single dosesystemic administration lies in the range of about 0.5 to 2.5 mg perrat, or 1-50 or 5-25 mg/kg body weight. It will be appreciated thatdetermining preferred dosages for treating individuals can be determinedby routine experimentation.

[0140] In related experiments, single doses (2.5 mg OP-1/rat)administered at 24 hr. post implant were evaluated at days 3, 5, 7, 9,11, 14, 21, 28 and 60 to determine the rate and quality of new bone(callus) formation. The animals were sacrificed on days 3, 5, 7, 9, 11,14, 21 and 28 and 60 after OP-1 administration and examined for new boneformation by biochemical assays and by histology. Data show good boneformation by 1 week and that bone formation follows the same biology oflocally provided protein, namely a conserved progression through thecommitment steps evidencing true bone formations; including recruitmentof mesenchymal progenitor cells, proliferation of chondrocytes, matrixdeposition, osteoblast recruitment and proliferation; remodeling, andbone marrow formation.

[0141] In a separate experiment, the potential ofsystemically-administered morphogenic proteins to initiate new boneformation at local ectopic sites where collagen carrier alone wasimplanted. Approximately 25 mg each of bovine bone-derived insolubletype I collagen was implanted at subcutaneous (left and right side ofthoracic region) and intramuscular (left and right thigh skeletalmuscle) sites of 6-8 week old Long-Evans male rats. Four rats per groupwere used. OP-1 (500 ul) was given intravenously through the tail veinat concentrations of 0.05, 0.5, 1.25, and 2.5 mg/rat. Groups receivingbuffer with a collagen implant and systemic OP-1 with a mock implantwere used as controls. Osteogenic activity was determined by histology,alkaline phosphatase activity, and calcium content at day 12 after OP-1administration.

[0142]FIG. 1 shows that intravenously administered OP-1 induces new boneformation in a dose-dependent manner as measured by alkaline phosphateactivity, calcium content, and histologic examination of implantsharvested on day 12 after OP-1 administration. Maximal bone-formingactivity was observed when a dose of 2.5 mg OP-1/rat was used. About 30%maximal activity was observed when 0.05 mg OP-1/rat was used. A singleadministration of 2.5 mg OP-1 given in one injection (24 hourspost-implant), produced comparable bone-forming activity as 5 injectionsadministered at 0, 8, 24, 48, and 72 hours after implantation producedcomparable bone-forming activity. Importantly, the amount of OP-1administered systemically to effect maximal bone formation is about1000-fold higher than the amount necessary to induce bone formation whenthe collagen/OP-1 device is used to induce bone locally. Collagenimplants at intramuscular sites appear to work better than those atsubcutaneous sites, as responding cells and vascular components arereadily available at intramuscular sites.

EXAMPLE 5

[0143] The following example provides a protocol for comparativemeasurement of bone formation where protein is provided by any systemicroute, namely: i.v., intraperitoneal or by oral administration. Liquidsolutions of OP-1 were used for all administration routes as follows.

[0144] Intravenous and Intraperitoneal: 500 μg (mature equivalent) doseadministered at 0, 6, 24, 48 and 72h (in 500 μl volume) after collagenimplant. Oral administration: soluble and mature OP-1, administered asone time doses of 2.5 mg in 1-2 ml at 24 h. With respect to i.v. andoral administration mOP-1 is taken in sodium acetate buffer for i.v.administration and in 0.1% casein in PBS for oral administration. OP-1is completely soluble in 0.1% casein. The day of OP-1 administration isconsidered as day 0 and implants harvested on day 12. The systemic OP-1induced osteogenesis in the collagen implant then is evaluated based onthe specific activity of alkaline phosphatase, calcium content andhistology.

[0145] Alternative Oral Administration Protocol: 2.5 mg protein (matureOP-1) solubilized in dilute methylene chloride and confined polymersolution (i.e., FA:SA, 20:80) and encapsulated by phase inversion.

[0146] Results: It is anticipated that all three routes will induceosteogenesis at the test implant site. The methodology provides meansfor assaying preferred dosages and formulations for a desiredadministration route.

[0147] Histochemical and biochemical analyses show that bone formationinduced by systemically-administered OP-1 in local collagen implantsundergoes a similar cascade of cellular events as that induced by localOP-1/collagen implants.

EXAMPLE 6

[0148] This example demonstrates the ability of other systemicallyadministered morphogenic proteins to induce local bone formation. Usingthe protocols described above, recombinant BMP-2, CDMP-1 and CDMP-2 wereevaluated. In the assay OP-1, BMP-2 (at 1.25 mg) and CDMP-1 and CDMP-2(at 2.5 mg) were administered systemically (tail vein, 500 μl) at 24 hrsafter collagen implant. The day of protein administration was consideredas day 0 and implants were harvested on day 12.

EXAMPLE 7

[0149] This example evaluates the effect of the age of the animal onosteogenesis in the collagen implants at ectopic sites and the abilityof systemically administered morphogenic protein to accelerate orimprove healing in adult rats. Typically, juvenile rats heal faster thanaged rats. For example, when hair line fractures are induced in juvenilerats (e.g., 1 month old), callus formation can be seen by one week, andcomplete healing occurs by three weeks. By contrast, in adult rats (24months old) formation takes two weeks and complete healing requires 6-12weeks.

[0150] Long-Evans or Fisher rats at 1, 3, 6, 12 and 24 months old weresubjected to subcutaneous and intramuscular implants as detailed above.The implants consisted of bovine collagenous matrix (approximately 25mg) only. Four implants were made per rat, with four rats per group.Mature OP-1 was administered at 0, 6, 24, 48 and 72 hrs at 500 μg per500 μl in acetate buffer. The day of OP-1 administration is consideredas Day 0. Animals were evaluated at selected time points within thefollowing day range: days 3, 5, 7, 9, 11, 14, 21, 28 and 60.

[0151] Results: Systemically administered morphogenic protein resultedin faster callus and bone formation in older rats as compared withcontrols. The data demonstrates the utility of systemic administrationas a means for enhancing bone repair in adults or any individuals havinga reduced capacity for bone healing and who experience refractoryhealing. Such individuals have a reduced or delayed ability to repairfractures either because of a lack of precursor cells, poor vascularity,reduced inductive signals or the like.

[0152] A comparison of OP-1 effects with related members of themorphogen family shows that OP-1 is more potent than BMP-2, CDMP-1(GDF-5), and CDMP-2 (GDF-6) in inducing osteogenesis upon systemicadministration as measured by calcium content and histology performed onday 12 implants. The effect on osteogenesis at local collagen implantsites in animals given OP-1 systemically was independent of age. Resultsare shown in FIGS. 3A and 3B. As shown in FIG. 3B there was some delayin the rate of bone remodeling and mineralization as determined bycalcium content.

EXAMPLE 8

[0153] This example demonstrates the ability of systemicallyadministered morphogenic protein to induce osteogenesis in a criticalsize ulna segmental defect in an art-recognized canine model. A criticalsize bone defect is incapable of spontaneous healing, and demonstratesthe ability of morphogenic protein to induce new tissue formationpermissive locus in a typically nonregenerating tissue. Briefly, adultmale mongrel dogs, supplied by authorized kennels, are utilized becausetheir well-known bone repair and remodeling characteristics Animalspreferably are at least two years old and weigh from 40 to 50 pounds,with special attention was paid in selecting animals of uniform size andweight to limit the variability in bone geometry and loading. Bilateral2.5 cm ulna segmental defects are created in individual dogs usingstandard surgical procedures. All right side defects are left untreated,all left side defects receive the standard morphogenic protein deviceusing, for example, recombinant human morphogenic protein-1 (rhOP-1)admixed with bovine bone Type I collagen matrix at a ratio of 2.5 mgrhOP-1 per gram of collagen matrix. Mature or soluble morphogenicprotein is administered in dose ranges of 0.5 mg-5 mg (500-1,000 ml),administered as a single bolus at least at 24 hrs, 48 hrs, or 72 hrspost surgery, or as a series of five separate injections as describedabove. Control groups include no systemically administered morphogenicprotein, a mock implant; and animals subjected to a single test defect,treated either with a standard device or systemically administeredprotein. Biweekly radiographs are taken to study the progression ofhealing and graded on a 0-6 scale. At sacrifice, all ulnae are retrieveden bloc, and those that are healed sufficiently upon manual manipulationare mechanically tested in torsion. Segments then are evaluated byhistology for tissue response, bone architecture and remodeling, andquality and amount of new bone formation and healing. It is anticipatedthat systemically administered morphogenic protein will induceosteogenesis sufficient to repair the ulna defect.

EXAMPLE 9

[0154] Provided below is a standard animal pulmonary fibrosis model andcan be used to demonstrate the ability of systemically administeredmorphogens to repair damaged lung tissue. The example essentiallyfollows the methods described in Haston et al. (1996) Cancer Research56:2596-2601; Weinbach et al. (1996) Cancer Research 56:5659-5665;Harrison et al. (1988) J. Pharmacol. Exp. Thr. 247:1052-1058.

[0155] Pulmonary fibrosis is a potentially lethal, chronic response ofthe lung to injury caused by bleomycin (BLM), a highly usefulantineoplastic agent that lacks substantial bone morrow toxicity. Thehallmark of this disorder is characterized by an increased deposition ofextracellular matrix proteins in the alveolar wall, notably collagen,which compromises pulmonary function.

[0156] In the assay, lung damage is induced in commercially availablelaboratory mice with BLM injections, typically 100, 300, 400 or 500mg/kg. The agent can be administered by interperitoneally orsubcutaneous injections, or by means of an implanted pump. Preferably,the C₅₇BL/6J mouse strain is utilized (Jackson Laboratoryies), which hasa fibrosis-prone phenotype, e.g., histological lesions and increasedpulmonary OH-praline content characteristic of pulmonary fibrosis. Afibrosis-resistant phenotype strain, such as C₃hf/kam can be used as acontrol strain.

[0157] In the example, buffer alone, mature morphogen or the solublecomplex form are administered in one or more of the following protocols:

[0158] A. Single bolus (0.05, 0.5, 1.0, or 2.5 mg/500 ml) administeredin 500 μl volumes at 0, 6, 12, 24, 48 or 72 hours after BLM injection.

[0159] B. Multiple injections up to a total of (0.05, 0.5, 1.0 or 2.5mg), administered in 500 μl volumes at time 0, 6, 24, 48, and 72 hours.

[0160] The animals are sacrificed by cervical dislocation at 8, 10 or 12weeks or when in respiratory distress as indicated by an elevatedbreathing rate. The lungs then are evaluated by histology and OH-prolinecontent using standard methodologies.

[0161] The results are expected to demonstrate that, in the absence ofmorphogenic protein, fibrosis is induced by moderate doses of BLM(cumulative dose, 300 or 400 mg/kg). In contrast mice treated withsystemically administered morphogens show substantially reduced or nofibrotic lesions of fibrosis and these mice tolerate higher i.p.-inoculated doses of BLM.

EXAMPLE 10

[0162] Myocardial infarcts heal by scarring because myocardium cannotregenerate. This assay demonstrates the ability of systemicallyadministered morphogens to regenerate new contractile tissue, andsubstantially restore function. The assay follows the protocol of Murryet al. (1996) J. Clin. Invest 98(11):2512-2523.

[0163] Hearts of adult inbred rats are injured by a standardfreeze-thaw, methodology. Specifically, a 1-cm-diameter aluminum rod,precooled with liquid nitrogen, is placed in direct contact with theanterior left ventricle for 15 seconds. Freeze-thaw reproducibly causesa disc-shaped region of coagulation necrosis, ˜1 cm in diameter,extending ˜2 mm into the myocardium. Morphogen or saline buffer alone isadministered at time t=0, 6, 24, 48 and 72 hours after injury. Morphogencan be administered as a single bolus or by multiple injections, asdescribed in Example 10. By 1 week, multimucleated myotubes are evidentin repairing tissue. At 2 wks, satellite stem cells are evident. By 7weeks β-MHC expression is detected.

[0164] At 1, 2 and 7 weeks and myocardium tissue formation evaluated byhistology and standard biochemical marker assays, including evidence ofβ-MHC expression.

EXAMPLE 11

[0165] Morphogen Expression in Regenerating Liver Tissue FollowingToxin-Induced Tissue Damage

[0166] Hepatic tissue repair following toxic agent-induced damagedtissue involves proliferation and differentiation of hepatocyteprecursor cells. This tissue reparation apparently mimics the tissuemorphogenesis cascade that occurs during embryogenesis (Fausto, etal.(1989) Lab. Investigation 60:4-13). As demonstrated in the examplebelow, systemically administered morphogen can enhance hepatic tissueregeneration following galactosamine or carbon tetrachloride(CCl₄)-induced liver damage. Experiments are performed essentially asdescribed in Kuhlmann et al., (1980) Virchows Arch 387:47-57, thedisclosure of which is incorporated herein by reference.galactosamine-HCl 0.75 g/.kg body weight on day 0. Morphogen isadministered In this experiment, male rats were provided with a singleintraperitoneal injection of systemically (e.g., according to thefollowing protocol: 0.5, 1.0 or 2.5 mg m OP-1 in 500 μg buffer (acetateor PBS), at 6, 12, 24 or 48 hours post toxin injection. Animals aresacrificed on days 3, 5, 12, 20, and evaluated by histology.

EXAMPLE 12

[0167] Morphogen-Induced Liver Regeneration

[0168] This Example demonstrates the ability of systemicallyadministered morphogen to regenerate new liver tissue following apartial hepatectomy.

[0169] While hepatocytes have a remarkable capacity to undergocompensatory growth following tissue loss, the reparative properties ofliver differ significantly from embryonic morphogenesis. Specifically,following a partial hepatectomy wherein a liver lobe is partially orcompletely removed, the remaining intact lobes grow rapidly and doublein weight due to the ability of the differentiated hepatocytes in theintact lobe to undergo limited proliferation. However, the excised lobeitself is not regenerated. The following example demonstrates theability of morphogens to regenerate lost hepatic tissue following apartial hepatectomy, including regenerating the excised tissue lobe. Theprotocol described below is a variation on a standard partialhepatectomy protocol, described, for example, by Higgins et al. (1931)Arch. Pathol. 12:136-202 and Braun et al. (1989) PNAS 86:1558-1562, thedisclosures of which are incorporated herein by reference.

[0170] Growing rats or aged rats are anesthetized by using ketamine. Twoof the liver lobes (left and right) are cut out (approximately ⅓ of thelobe). The wound is closed using standard surgical procedures.Morphogen, e.g., purified recombinant human OP-1, mature form, isadministered systemically intravenously (i.e.) orp. (interitoneally).OP-1 (mature or soluble) is injected at 0.5, 1.0 or 2.5 mg protein in500 μl buffer (acetate or PBS), at times 12, 24, 48 or 72 hours postsurgery. Placebo samples are injection buffer without morphogen.Following surgery the rats are allowed to eat normal food and drink tapwater.

[0171] After 12 days, the rats are sacrificed and liver regeneration isobserved visually. OP-1 injected group(s) will show complete livertissue regeneration including reformation of the excised lobe tissue,and show no substantial sign of any cut in the liver. By contrast, inthe control group into which only PBS is injected, the excised lobetissue is not substantially regenerated. The original incision typicallyremains in this sample.

[0172] In a variation on this example, morphogen is administered as aseries of injections over a period of 3-10 days following surgery.

EXAMPLE 13

[0173] Morphogen Treatment of Oral Mucositis

[0174] Oral mucositis involves ulcerations of the mouth as a consequenceof, e.g., radiation therapy or chemotherapy. The course of ulcerativemucositis may be divided into a destructive phase and a healing phase.Since the cells of the basal layer of the oral epithelium divide at arapid rate, they are susceptible to the antimitogenic and toxic effectsof chemotherapy. As a result, atrophic changes occur which then arefollowed by ulceration. This constitutes the destructive phase.Following ulcer formation, the lesions slowly resolve during the healingphase.

[0175] The example below demonstrates the efficacy of systemicallyadministered morphogen in protecting the oral mucosa from oral mucositisin a hamster model, including both inhibiting ulceration and enhancingregeneration of ulcerated tissue. Systemic administration eliminatesproblems associated with maintaining topically applied morphogen at adefect locus.

[0176] Details of the protocol can be found in Sonis, et al., (1990)Oral Surg. Oral Med. Oral Pathol 69: 437-443, the disclosure of which isincorporated herein by reference.

[0177] Briefly, golden syrian hamsters (6-8 wks old, Charles RiverLaboratories, Wilmington, Mass.) are divided into test groups: a placebo(e.g., saline) control, and a morphogen low dose group (100 ng) and amorphogen high dose group (1 μg), Groups 2 and 3, respectively.Additional groups can modulate the number of injections morphogen is(single bolus vs. multiple administration). Each group contain the samenumber of animals. Beginning on day 0 and continuing through day 5morphogen is administered systemically (mature form) (oral, or ip, oriv; (tail vein)). On day 3, all groups begin the mucositis-inductionprocedure. 5-fluorouracil is injected intraperitoneally on days 3 (60mg/kg) and 5 (40 mg/kg). On day 7, the right buccal pouch mucosa issuperficially irritated with a calibrated 18 gauge needle. In untreatedanimals, severe ulcerative mucositis is induced in at least 80% of theanimals by day 10.

[0178] On day 12, two animals in each group are sacrificed forhistological studies. The right buccal pouch mucosa and underlyingconnective tissue are dissected and fixed in 10% formalin using standarddissection and histology procedures. The specimens are mounted inparaffin and prepared for histologic examination. Sections then arestained with hematoxylin and eosin and are examined blindly by threeoral pathologists with expertise in hamster histology and scored blindagainst a standard mucositis panel. The extent of atrophy, cellularinfiltration, connective tissue breakdown, degree of ulceration andepithelialization are assessed.

[0179] Based on histology, administered morphogen inhibits lesionformation significantly in a dose-dependent manner. By contrast,significant tissue necrosis, indicated by the dark regions in thetissue, and ulceration, indicated by the light globular areas in thetissue, is evident in untreated pouches. The morphogen-treated tissueshows healthy tissue with no necrosis and little or no ulceration.Single administration of high doses are contemplated to be substantiallyas effective as multiple injection doses.

[0180] The method of the invention allows an individual at risk to takethe protein prophylactically, concurrent with a cancer therapy regimenallowing morphogen to be present in the system when at the time theinitial oral mucositis defect occurs.

EXAMPLE 14

[0181] Morphogen Treatment of Duodenal Ulcer Formation

[0182] The following example provides a rat model for demonstrating theefficacy of systemically administering morphogen in treating duodenalulcers. A detailed description of the protocol is provided in Pilan etal., (1985) Digestive Diseases and Sciences 30:240-246, the disclosureof which is incorporated. herein by reference. Briefly, Sprague-Dawleyfemale rats (e.g., Charles River Laboratories, 150-200 grams) receivethe duodenal ulcerogen cysteamine-HCl at a dose of 25-28 milligrams (mg)per 100 grams (gm) of body weight orally by intragastric gavage 3 timeson the same day. Additionally, cortisol is administered subcutaneouslyto each rat at a single dose of 5 mg of cortisol to 100 gm of bodyweight to decrease the mortality resulting from the administration ofthe cysteamine-HCl.

[0183] Three days after administration of the cysteamine-HCl, ratshaving penetrating and perforating duodenal ulcers are identified bystandard laparotomy and randomized into control and morphogen-treatedgroups.

[0184] The rats of Group 1, all of which have ulcers, receive nomorphogen and are treated only with saline. The rats of Group 2 each ofwhich also have ulcers, receive 50-100 ng of morphogen per 100 gm ofbody weight administered i.v. (k.l vein) or i.p. or by oraladministration. Group 3 rats, all of which have ulcers, receive 200-500ng of morphogen per 100 gm of body weight. Treatments also can be asingle bolus or a series of multiple injections, as described in Example10. Animals are sacrificed on day 21, and the ulcers measured andhistologic sections taken.

[0185] Histology of duodenal sections from morphogen-treated animalsshows healed ulcers with prominent and dense granulation tissue andpartial or complete re-epithelialization, demonstrating that oraladministration of morphogen can significantly accelerate the healing ofulcers of the GI tract. Moreover, treatment with morphogen before orconcomitantly with ulceration also inhibits ulcer formation.

EXAMPLE 15

[0186] Morphogen Treatment of Retinal Disorders

[0187] Systemically administered morphogen also can be used to treatretinal disorders, particularly for treatment of macular degenerationand holes, where it is anticipated to promote healing and significantlyimprove vision. The treatment also can be used on retinal holes, tears,and detachment. These disorders are characterized by loss of visualacuity.

[0188] Eyes to be treated with morphogen undergo complete pre- andpost-operative ocular examination including visual acuity testing,intraocular pressure measurements, slit-lamp bio-microscopy, andbinocular indirect ophthalmoscopy. Surgical strategy will vary dependentupon the exact vitreoretinal anatomy, as is known in the art. Generally,all vitrectomies are performed with standard three-port instrumentationusing standard procedures. Vitrectomy is performed prior to morphogenadministration.

[0189] Morphogen is administered systemically (ip., iv) followingvitrectomy recording to the protocol outlined in Example 10, or avariation thereof. Animals are sacrificed at 1 week, 2 weeks, 7 weeksand evaluated by histology. Systemically administered morphogeneliminates the need to provide morphogen by local injection and isanticipated to promote reattachment of the retina and regeneration oflost retinal tissues at least as well as locally provided protein. Inaddition, systemic administration of morphogen should inhibitreproliferation of fibrovascular tissue, and inhibit neovascularization.

[0190] Morphogen similarly can be administered to treat macular holes.Such treatment is expected to provide improvement of vision and healingby decreasing the thickness of the edge of the hole.

EXAMPLE 16

[0191] This example demonstrates the ability of systemicallyadministered osteogenic protein to correct an osteochondral or chondraldefect, using an art-recognized animal model (dog). Specifically, astudy using a standard dog osteochondral plug defect model is conductedas described below. Briefly, full thickness defects 5 mm in diameter andextending 6 mm into the subchondral bone are created bilaterally on themedial femoral condyle of 4 adult mongrel dogs using standard surgicalprocedures and animals supplied by authorized kennels. The left sidedefects receive standard osteogenic device and the right side defect isleft untreated. Systemic administration assays are carried out as forthe critical site defect in Example 8 above, with appropriate controls,including mock implants. That is, a range of useful dosages areevaluated (0.5 mg-5 mg) administered as a single bolus at least at 24,48, or 72 hrs post surgery, or administered in multiple separateinjections.

[0192] Osteochondral and chondral healing is evaluated grossly andhistologically using routine protocols, including radiographs toevaluate healing. At twelve weeks post-operative each animal issacrificed by an intravenous barbiturate overdose. Both right and leftdistal femurs are harvested en bloc and kept in cool saline until grossgrading and microphotography are completed. The specimens are placed in4% paraformaldehyde fixative, labeled with all necessaryidentifications, and stored at 4° C. until evaluated.

[0193] For histologic evaluation, the individual specimens are fixed byimmersion in 4% paraformaldehyde solution and evaluated using standardprocedures. In addition, using routine procedures, tissue typinganalysis is performed in order to characterize the collagen type andpercent tissue composition. Non-decalcified sections, one from eachspecimen, stained with Safranin-O and Fast Green stains (to indicateglycosaminoglycan content in the matrix), also can be used.

[0194] Osteochondral and/or condral defects treated with systemicallyadministered osteogenic protein are anticipated to demonstrate boneand/or cartilage regeneration as the case may be, including appropriatechondrocyte and cartilage phenotype, including functional reparativearticular cartilage formation, as compared with defects treated with thestandard osteogenic device.

1 9 1 1822 DNA Homo sapiens CDS (49)..(1341) “Morphogenic Protein OP-1”1 ggtgcgggcc cggagcccgg agcccgggta gcgcgtagag ccggcgcg atg cac gtg 57Met His Val 1 cgc tca ctg cga gct gcg gcg ccg cac agc ttc gtg gcg ctctgg gca 105 Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu TrpAla 5 10 15 ccc ctg ttc ctg ctg cgc tcc gcc ctg gcc gac ttc agc ctg gacaac 153 Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn20 25 30 35 gag gtg cac tcg agc ttc atc cac cgg cgc ctc cgc agc cag gagcgg 201 Glu Val His Ser Ser Phe Ile His Arg Arg Leu Arg Ser Gln Glu Arg40 45 50 cgg gag atg cag cgc gag atc ctc tcc att ttg ggc ttg ccc cac cgc249 Arg Glu Met Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu Pro His Arg 5560 65 ccg cgc ccg cac ctc cag ggc aag cac aac tcg gca ccc atg ttc atg297 Pro Arg Pro His Leu Gln Gly Lys His Asn Ser Ala Pro Met Phe Met 7075 80 ctg gac ctg tac aac gcc atg gcg gtg gag gag ggc ggc ggg ccc ggc345 Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Gly Gly Gly Pro Gly 8590 95 ggc cag ggc ttc tcc tac ccc tac aag gcc gtc ttc agt acc cag ggc393 Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gln Gly 100105 110 115 ccc cct ctg gcc agc ctg caa gat agc cat ttc ctc acc gac gccgac 441 Pro Pro Leu Ala Ser Leu Gln Asp Ser His Phe Leu Thr Asp Ala Asp120 125 130 atg gtc atg agc ttc gtc aac ctc gtg gaa cat gac aag gaa ttcttc 489 Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe Phe135 140 145 cac cca cgc tac cac cat cga gag ttc cgg ttt gat ctt tcc aagatc 537 His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys Ile150 155 160 cca gaa ggg gaa gct gtc acg gca gcc gaa ttc cgg atc tac aaggac 585 Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg Ile Tyr Lys Asp165 170 175 tac atc cgg gaa cgc ttc gac aat gag acg ttc cgg atc agc gtttat 633 Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg Ile Ser Val Tyr180 185 190 195 cag gtg ctc cag gag cac ttg ggc agg gaa tcg gat ctc ttcctg ctc 681 Gln Val Leu Gln Glu His Leu Gly Arg Glu Ser Asp Leu Phe LeuLeu 200 205 210 gac agc cgt acc ctc tgg gcc tcg gag gag ggc tgg ctg gtgttt gac 729 Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu Val PheAsp 215 220 225 atc aca gcc acc agc aac cac tgg gtg gtc aat ccg cgg cacaac ctg 777 Ile Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His AsnLeu 230 235 240 ggc ctg cag ctc tcg gtg gag acg ctg gat ggg cag agc atcaac ccc 825 Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Ser Ile AsnPro 245 250 255 aag ttg gcg ggc ctg att ggg cgg cac ggg ccc cag aac aagcag ccc 873 Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn Lys GlnPro 260 265 270 275 ttc atg gtg gct ttc ttc aag gcc acg gag gtc cac ttccgc agc atc 921 Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe ArgSer Ile 280 285 290 cgg tcc acg ggg agc aaa cag cgc agc cag aac cgc tccaag acg ccc 969 Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser LysThr Pro 295 300 305 aag aac cag gaa gcc ctg cgg atg gcc aac gtg gca gagaac agc agc 1017 Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu AsnSer Ser 310 315 320 agc gac cag agg cag gcc tgt aag aag cac gag ctg tatgtc agc ttc 1065 Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr ValSer Phe 325 330 335 cga gac ctg ggc tgg cag gac tgg atc atc gcg cct gaaggc tac gcc 1113 Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu GlyTyr Ala 340 345 350 355 gcc tac tac tgt gag ggg gag tgt gcc ttc cct ctgaac tcc tac atg 1161 Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu AsnSer Tyr Met 360 365 370 aac gcc acc aac cac gcc atc gtg cag acg ctg gtccac ttc atc aac 1209 Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val HisPhe Ile Asn 375 380 385 ccg gaa acg gtg ccc aag ccc tgc tgt gcg ccc acgcag ctc aat gcc 1257 Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr GlnLeu Asn Ala 390 395 400 atc tcc gtc ctc tac ttc gat gac agc tcc aac gtcatc ctg aag aaa 1305 Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val IleLeu Lys Lys 405 410 415 tac aga aac atg gtg gtc cgg gcc tgt ggc tgc cactagctcctcc 1351 Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 420 425430 gagaattcag accctttggg gccaagtttt tctggatcct ccattgctcg ccttggccag1411 gaaccagcag accaactgcc ttttgtgaga ccttcccctc cctatcccca actttaaagg1471 tgtgagagta ttaggaaaca tgagcagcat atggcttttg atcagttttt cagtggcagc1531 atccaatgaa caagatccta caagctgtgc aggcaaaacc tagcaggaaa aaaaaacaac1591 gcataaagaa aaatggccgg gccaggtcat tggctgggaa gtctcagcca tgcacggact1651 cgtttccaga ggtaattatg agcgcctacc agccaggcca cccagccgtg ggaggaaggg1711 ggcgtggcaa ggggtgggca cattggtgtc tgtgcgaaag gaaaattgac ccggaagttc1771 ctgtaataaa tgtcacaata aaacgaatga atgaaaaaaa aaaaaaaaaa a 1822 2 431PRT Homo sapiens 2 Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His SerPhe Val Ala 1 5 10 15 Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala LeuAla Asp Phe Ser 20 25 30 Leu Asp Asn Glu Val His Ser Ser Phe Ile His ArgArg Leu Arg Ser 35 40 45 Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Leu SerIle Leu Gly Leu 50 55 60 Pro His Arg Pro Arg Pro His Leu Gln Gly Lys HisAsn Ser Ala Pro 65 70 75 80 Met Phe Met Leu Asp Leu Tyr Asn Ala Met AlaVal Glu Glu Gly Gly 85 90 95 Gly Pro Gly Gly Gln Gly Phe Ser Tyr Pro TyrLys Ala Val Phe Ser 100 105 110 Thr Gln Gly Pro Pro Leu Ala Ser Leu GlnAsp Ser His Phe Leu Thr 115 120 125 Asp Ala Asp Met Val Met Ser Phe ValAsn Leu Val Glu His Asp Lys 130 135 140 Glu Phe Phe His Pro Arg Tyr HisHis Arg Glu Phe Arg Phe Asp Leu 145 150 155 160 Ser Lys Ile Pro Glu GlyGlu Ala Val Thr Ala Ala Glu Phe Arg Ile 165 170 175 Tyr Lys Asp Tyr IleArg Glu Arg Phe Asp Asn Glu Thr Phe Arg Ile 180 185 190 Ser Val Tyr GlnVal Leu Gln Glu His Leu Gly Arg Glu Ser Asp Leu 195 200 205 Phe Leu LeuAsp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu 210 215 220 Val PheAsp Ile Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg 225 230 235 240His Asn Leu Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Ser 245 250255 Ile Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn 260265 270 Lys Gln Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe275 280 285 Arg Ser Ile Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn ArgSer 290 295 300 Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Asn ValAla Glu 305 310 315 320 Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys LysHis Glu Leu Tyr 325 330 335 Val Ser Phe Arg Asp Leu Gly Trp Gln Asp TrpIle Ile Ala Pro Glu 340 345 350 Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly GluCys Ala Phe Pro Leu Asn 355 360 365 Ser Tyr Met Asn Ala Thr Asn His AlaIle Val Gln Thr Leu Val His 370 375 380 Phe Ile Asn Pro Glu Thr Val ProLys Pro Cys Cys Ala Pro Thr Gln 385 390 395 400 Leu Asn Ala Ile Ser ValLeu Tyr Phe Asp Asp Ser Ser Asn Val Ile 405 410 415 Leu Lys Lys Tyr ArgAsn Met Val Val Arg Ala Cys Gly Cys His 420 425 430 3 102 PRT ArtificialSequence Description of Artificial Sequence OPX 3 Cys Xaa Xaa His GluLeu Tyr Val Ser Phe Xaa Asp Leu Gly Trp Xaa 1 5 10 15 Asp Trp Xaa IleAla Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly 20 25 30 Glu Cys Xaa PhePro Leu Xaa Ser Xaa Met Asn Ala Thr Asn His Ala 35 40 45 Ile Xaa Gln XaaLeu Val His Xaa Xaa Xaa Pro Xaa Xaa Val Pro Lys 50 55 60 Xaa Cys Cys AlaPro Thr Xaa Leu Xaa Ala Xaa Ser Val Leu Tyr Xaa 65 70 75 80 Asp Xaa SerXaa Asn Val Ile Leu Xaa Lys Xaa Arg Asn Met Val Val 85 90 95 Xaa Ala CysGly Cys His 100 4 97 PRT Artificial Sequence Description of ArtificialSequence Generic Sequence 7 4 Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly TrpXaa Xaa Trp Xaa Xaa Xaa 1 5 10 15 Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr CysXaa Gly Xaa Cys Xaa Xaa Pro 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa AsnHis Ala Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Cys Cys Xaa Pro 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa LeuXaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Val Xaa Leu Xaa Xaa Xaa Xaa XaaMet Xaa Val Xaa Xaa Cys Xaa Cys 85 90 95 Xaa 5 102 PRT ArtificialSequence Description of Artificial Sequence Generic Sequence 8 5 Cys XaaXaa Xaa Xaa Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa 1 5 10 15 XaaXaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly 20 25 30 XaaCys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala 35 40 45 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 XaaCys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa 65 70 75 80Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val 85 90 95Xaa Xaa Cys Xaa Cys Xaa 100 6 97 PRT Artificial Sequence Description ofArtificial Sequence Generic Sequence 9 6 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Pro Xaa Xaa Xaa Xaa Xaa XaaXaa Cys Xaa Gly Xaa Cys Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 85 90 95 Xaa 7 102 PRTArtificial Sequence Description of Artificial Sequence Generic Sequence10 7 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 510 15 Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Gly 2025 30 Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 3540 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 5055 60 Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa 6570 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa85 90 95 Xaa Xaa Cys Xaa Cys Xaa 100 8 5 PRT Artificial SequenceDescription of Artificial Sequence Peptide 8 Cys Xaa Xaa Xaa Xaa 1 5 9 5PRT Artificial Sequence Description of Artificial Sequence Peptide 9 CysXaa Xaa Xaa Xaa 1 5

What is claimed is:
 1. A method for evaluating the morphogenic activityof a candidate morphogenic protein or analog thereof, the methodcomprising the steps of: (a) creating a local permissive defect site ina mammal, (b) administering a said candidate morphogenic protein oranalog systemically to said mammal, and (c) measuring the ability ofcandidate protein or analog to induce new tissue formation at saiddefect site.
 2. The method of claim 1 wherein said candidate morphogenicprotein or analog is administered at a site distal to said defect site.3. A method for evaluating an optimal dosage of a candidate morphogenicprotein or analog thereof for administering to a mammal, the methodcomprising the steps of: (a) creating a local permissive defect site ina mammal, and (b) administering a said candidate morphogenic protein oranalog systemically to said mammal, and (c) measuring the ability ofcandidate protein or analog to induce new tissue formation at saiddefect site.
 4. The method of claim 3 wherein said protein or analog isadministered at a site distal to said locus.
 5. The method of claim 1 or3 wherein said defect locus occurs in skeletal, lung, cardiac, liver,neural, pancreas, uterine, or thyroid tissue.
 6. The method of claim 1or 3 wherein said defect locus occur in renal tissue.
 7. The method ofclaim 1 or 3 wherein said defect locus occurs in dental or periodontaltissue.
 8. The method of claim 1 or 3 wherein said mammal is aged. 9.The method of claim 1 or 3 wherein said mammal has a reduced capacity toinduce callus formation.
 10. The method of claim 1 or 3 wherein saidmammal is afflicted with impaired blood flow to the skeletalextremities.
 11. The method of claim 1 or 3 wherein said mammal has areduced capacity to induce an endogenous morphogenetic signal.
 12. Themethod of claim 1 or 3 wherein morphogenic protein or analog isadministered parenterally.
 13. The method of claim 12 whereinmorphogenic protein or analog is administered intravenously.
 14. Themethod of claim 1 or 3 wherein said morphogenic protein is administeredorally.
 15. The method of claim 1 wherein said morphogenic protein oranalog is administered to said mammal at a time when mesenchymalprogenitor cells are accessible to said defect locus.
 16. The method ofclaims 1, 3 or 4 wherein said morphogenic protein or analog isadministered at least six hours after the creation of said defect. 17.The method of claim 1 or 4 wherein said morphogenic protein or analog isadministered at least 24 hours after the creation of said defect. 18.The method of claim 1 or 4 wherein said morphogenic protein or analog isadministered at least 72 hours after the creation of said defect. 19.The method of claims 1, 3 or 4 wherein said morphogenic protein oranalog is administered to said mammal after the initiation of fibro sisat said defect locus.
 20. The method of claims 1, 3 or 4 wherein saidmorphogenic protein or analog is administered in aqueous solution. 21.The method of claim 8 wherein said mammal is a steroidal drug user. 22.The method of claim 8 wherein said mammal is aged, obese, hypertensive,or afflicted with osteopenia or diabetes.
 23. The method of claim 1 or 3wherein said morphogenic protein is selected from the group consistingof: OP1; OP2, OP3, BDP2; BMP3; BMP4; BMP5; BMP6; BMP9; BMP-10, BMP-11,BMP-12, BMP-15, BMP-3b, DPP; Vg1; Vgr; 60A protein; GDF-1; GDF-3, GDF-5,GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11; and morphogenically activeamino acid sequence variants thereof.
 24. The method of claim 1 or 3wherein said morphogenic protein is selected from the group consistingof: OP1; OP2, BMP2; BMP4; BMP5; BMP6; and morphogenically active aminoacid sequence variants thereof.
 25. The method of claim 1 or 3 whereinsaid morphogenic protein is a morphogen, said morphogen comprising anamino acid sequence having at least 70% homology within the C-terminal102-106 amino acids, including the conserved seven cysteine domain, ofhuman OP1.
 26. The method of claim 1 or 3 wherein said morphogenicprotein is OP1.
 27. The method of claim 1 or 3 wherein said morphogenicprotein is mature OP 1 solubilized in a saline solution.
 28. The methodof claim 1 or 3 wherein said morphogenic protein comprises an amino acidsequence defined by OPX (Seq. ID No.3); Generic Sequence 6 (Seq. IDNo.4, Generic Sequence 7 (Seq. ID No.5); Generic Sequence 8 (Seq. IDNo.6); or Generic Sequence 9 (Seq. ID No.7).
 29. A method for inducingnew tissue formation at a nonskeletal defect locus in a mammal, themethod comprising the step of administering morphogenic proteinsystemically to said mammal.
 30. The method of claim 29 wherein saidmorphogenic protein is administered at a site distal to said locus. 31.A method for enhancing the quantity or quality of callus formation at anmorphogenic defect locus in a mammal, the method comprising the step ofadministering morphogenic protein systemically to said mammal at a sitedistal to said locus.
 32. A method for enhancing the rate of tissuerepair at a local defect site in a mammal, the method comprising thestep of administering an morphogenic protein systemically to said mammalat a site distal to said locus.
 33. The method of claims 29, 31, or 32wherein said defect locus occur in lung, cardiac, liver, neural,pancreas, uterine, or thyroid tissue.
 34. The method of claims 29, 31,or 32 wherein said defect locus occur in renal tissue.
 35. The method ofclaims 29, 31, or 32 wherein said defect locus dontal or periodontaltissue.
 36. The method of claims 29, 31, or 32 wherein said mammal is ahuman.
 37. The method of claim 36 wherein said human has a reducedcapacity to induce callus formation.
 38. The method of claim 36 whereinsaid human is afflicted with impaired blood flow to the skeletalextremities.
 39. The method of claim 36 wherein said individual has areduced capacity to induce an endogenous morphogenetic signal.
 40. Themethod of claims 29, 31 or 32 wherein morphogenic protein isadministered parenterally.
 41. The method of claim 40 whereinmorphogenic protein is administered intravenously.
 42. The method ofclaims 29, 31, or 32 wherein said morphogenic protein is administeredorally.
 43. The method of claim 29 wherein said morphogenic protein isadministered to said individual at a time when mesenchymal progenitorcells are accessible to said defect locus.
 44. The method of claims 29,31, or 32 wherein said morphogenic protein is administered at least sixhours after the creation of said defect.
 45. The method of claim 29 or32 wherein said morphogenic protein is administered at least 24 hoursafter the creation of said defect.
 46. The method of claim 29 or 32wherein said morphogenic protein is administered at least 72 hours afterthe creation of said defect.
 47. The method of claims 29, 31, or 32wherein said morphogenic protein is administered to said mammal afterthe initiation of fibrosis at said defect locus.
 48. The method ofclaims 29, 31, or 32 wherein said morphogenic protein is administered inaqueous solution.
 49. The method of claim 36 wherein said human is asteroidal drug user.
 50. The method of claim 36 wherein said human isaged, obese, or afflicted with osteopenia or diabetes.
 51. The method ofclaims 29, 31, or 32 wherein said morphogenic protein is selected fromthe group consisting of: OP1; OP2, OP3, BMP2; BMP3; BMP4; BMP5; BMP6;BMP9; BMP-10, BMP-11, BMP-12, BMP-15, BMP-3b, DPP; Vg1; Vgr; 60Aprotein; GDF-1; GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10,GDF-11; and morphogenically active amino acid sequence variants thereof.52. The method of claims 29, 31, or 32 wherein said morphogenic proteinis selected from the group consisting of: OP1; OP2, BMP2; BMP4; BMP5;BMP6; and morphogenically active amino acid sequence variants thereof.53. The method of claims 29, 31, or 32 wherein said morphogenic proteinis a morphogen, said morphogen comprising an amino acid sequence havingat least 70% homology within the C-terminal 102-106 amino acids,including the conserved seven cysteine domain, of human OP1.
 54. Themethod of claims 29, 31, or 32 wherein said morphogenic protein is OP1.55. The method of claims 29, 31, or 32 wherein said morphogenic proteinis mature OP1 solubilized in a saline solution.
 56. The method of claims29, 31, or 32 wherein said morphogenic protein comprises an amino acidsequence defined by OPX (Seq. ID No.3); Generic Sequence 6 (Seq. IDNo.4, Generic Sequence 7 (Seq. ID No.5); Generic Sequence 8 (Seq. IDNo.6); or Generic Sequence 9 (Seq. ID No.7).
 57. A composition forsystemic administration of an morphogenic protein to a mammal, saidcomposition comprising morphogenic protein in an amount sufficient toinduce nonskeletal functional replacement tissue formation at a defectlocus.
 58. The composition of claim 57 wherein said compositioncomprises morphogenic protein dispersed in an aqueous solution.
 59. Thecomposition of claim 57 having a pH in the range of about 4-8.
 60. Thecomposition of claim 57 comprising physiologically buffered saline. 61.The composition of claim 57 wherein said morphogenic protein is providedat a concentration within the range of about 0.01-1000 mg/kg bodyweight.
 62. The composition of claim 57 comprising a formulation forparentral administration.
 63. The composition of claim 57 formulated fororal administration.
 64. The composition of claim 57 wherein saidmorphogenic protein is disposed in a biodegradable, biocompatiblemicrosphere.
 65. The composition of claim 57 comprising morphogenicprotein in a concentration range of about 0.01 g/ml-10.0 g/ml.
 66. Thecomposition of claim 57 comprising morphogenic protein in a conconcentration range of about 0.1 g/ml-1.0 g/ml.
 67. The composition ofclaim 57 wherein said morphogenic protein is associated with a moleculecompetent to enhance solubility of said protein in aqueous media. 68.The composition of claim 57 wherein said morphogenic protein comprisesof the soluble complex form of said protein.
 69. The composition ofclaim 57 further characterized as competent to enhance the rate oftissue formation at a local defect site.
 70. The composition of claim 57wherein said morphogenic protein is selected from the group consistingof: OP1; OP2, OP3, BMP2; BMP3; BMP4; BMP5; BMP6; BMP9; BMP-10, BMP-11,BMP-12, BMP-15, BMP-3b, DPP; Vg1; Vgr; 60A protein; GDF-1; GDF-3, GDF-5,GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11; and morphogenically activeamino acid sequence variants thereof.
 71. The composition of claim 57wherein said morphogenic protein is selected from the group consistingof: OP1; OP2, BMP2; BMP4; BMP5; BMP6; and morphogenically active aminoacid sequence variants thereof.
 72. The composition of claim 57 whereinsaid morphogenic protein is a morphogen, said morphogen comprising anamino acid sequence having at least 70% homology within the C-terminal102-106 amino acids, including the conserved seven cysteine domain, ofhuman OP1.
 73. The composition of claim 57 wherein said morphogenicprotein is OP
 1. 74. The composition of claim 57 wherein saidmorphogenic protein is mature OP1 solubilized in a saline solution. 75.The composition of claim 57 wherein said morphogenic protein comprisesan amino acid sequence defined by OPX (Seq. ID No.3); Generic Seq. 6(Seq. ID No.4); Generic Seq. 7 (Seq. ID No.5); Generic Seq. 8 (Seq. IDNo.6); or Generic Seq. 9 (Seq. ID No.7).
 76. A method for inducing boneor cartilage formation at a defect locus in a mammal, the methodcomprising the step of administering osteogenic protein systemically tosaid mammal.
 77. The method of claim 76 wherein said osteogenic proteinis administered at a site distal to said locus.
 78. A method forenhancing the quantity or quality of callus formation at an osteogenicdefect locus in a mammal, the method comprising the step ofadministering osteogenic protein systemically to said mammal at a sitedistal to said locus.
 79. A method for enhancing the rate of bone orcartilage repair at a local defect site in a mammal, the methodcomprising the step of administering an osteogenic protein systemicallyto said mammal at a site distal to said locus.
 80. The method of claims76, 78 or 79 wherein said defect locus defines a bone fracture.
 81. Themethod of claims 76, 78, or 79 wherein said defect locus defines avolume incapable of endogenous repair.
 82. The method of claims 76, 78,or 79 wherein said defect locus defines an osteochondral defect.
 83. Themethod of claims 76, 78, or 79 wherein said mammal is a human.
 84. Themethod of claim 83 wherein said human has a reduced capacity to inducecallus formation.
 85. The method of claim 83 wherein said human isafflicted with impaired blood flow to the skeletal extremities.
 86. Themethod of claim 83 wherein said individual has a reduced capacity toinduce an endogenous osteoinductive signal.
 87. The method of claims 76,78, or 79 wherein osteogenic protein is administered parenterally. 88.The method of claim 87 wherein osteogenic protein is administeredintravenously.
 89. The method of claims 76, 78, or 79 wherein saidosteogenic protein is administered orally.
 90. The method of claim 76wherein said osteogenic protein is administered to said individual at atime when mesenchymal progenitor cells are accessible to said defectlocus.
 91. The method of claims 76, 78, or 79 wherein said osteogenicprotein is administered at least six hours after the creation of saiddefect.
 92. The method of claim 76 or 79 wherein said osteogenic proteinis administered at least 24 hours after the creation of said defect. 93.The method of claim 76 or 79 wherein said osteogenic protein isadministered at least 72 hours after the creation of said defect. 94.The method of claims 76, 78, or 79 wherein said osteogenic protein isadministered to said mammal after the initiation of fibrosis at saiddefect locus.
 95. The method of claims 76, 78, or 79 wherein saidosteogenic protein is administered in aqueous solution.
 96. The methodof claim 83 wherein said human is a steroidal drug user.
 97. The methodof claim 83 wherein said human is aged, obese, or afflicted withosteopenia or diabetes.
 98. The method of claims 76, 78, or 79 whereinsaid osteogenic protein is selected from the group consisting of: OP1;OP2, OP3, BMP2; BMP3; BMP4; BMP5; BMP6; BMP9; BMP-10, BMP-11, BMP-12,BMP-15, BMP-3b, DPP; Vg1; Vgr; 60A protein; GDF-1; GDF-3, GDF-5, GDF-6,GDF-7, GDF-8, GDF-9, GDF-10, GDF-11; and morphogenically active aminoacid sequence variants thereof.
 99. The method of claims 76, 78, or 79wherein said osteogenic protein is selected from the group consistingof: OP1; OP2, BMP2; BMP4; BMP5; BMP6; and morphogenically active aminoacid sequence variants thereof.
 100. The method of claims 76, 78, or 79wherein said osteogenic protein is a morphogen, said morphogencomprising an amino acid sequence having at least 70% homology withinthe C-terminal 102-106 amino acids, including the conserved sevencysteine domain, of human OP1.
 101. The method of claims 76, 78, or 79wherein said osteogenic protein is OP1.
 102. The method of claims 76,78, or 79 wherein said osteogenic protein is mature OP 1 solubilized ina saline solution.
 103. The method of claims 76, 78, or 79 wherein saidosteogenic protein comprises an amino acid sequence defined by OPX (Seq.ID No.3); Generic Sequence 6 (Seq. ID No.4, Generic Sequence 7 (Seq. IDNo.5); Generic Sequence 8 (Seq. ID No.6); or Generic Sequence 9 (Seq. IDNo.7).
 104. A composition for systemic administration of an osteogenicprotein to a mammal, said composition comprising osteogenic protein inan amount sufficient to induce bone or cartilage formation at a skeletaldefect locus.
 105. The composition of claim 104 wherein said compositioncomprises osteogenic protein dispersed in an aqueous solution.
 106. Thecomposition of claim 104 having a pH in the range of about 4-8.
 107. Thecomposition of claim 104 comprising physiologically buffered saline.108. The composition of claim 104 wherein said osteogenic protein isprovided at a concentration within the range of about 0.01-1000 mg/kgbody weight.
 109. The composition of claim 104 comprising a formulationfor parentral administration.
 110. The composition of claim 104formulated for oral administration.
 111. The composition of claim 104wherein said osteogenic protein is disposed in a biodegradable,biocompatible microsphere.
 112. The composition of claim 104 comprisingosteogenic protein in a concentration range of about 0.01 g/ml-10.0g/ml.
 113. The composition of claim 104 comprising osteogenic protein ina con concentration range of about 0.1 g/ml-1.0 g/ml.
 114. Thecomposition of claim 104 wherein said osteogenic protein is associatedwith a molecule competent to enhance solubility of said protein inaqueous media.
 115. The composition of claim 104 wherein said osteogenicprotein comprises of the soluble complex form of said protein.
 116. Thecomposition of claim 104 further characterized as competent to enhancethe rate of bone formation at a local fracture site.
 117. Thecomposition of claim 104 wherein said osteogenic protein is selectedfrom the group consisting of: OP1; OP2, OP3, BMP2; BMP3; BMP4; BMP5;BMP6; BMP9; BMP-10, BMP-11, BMP-12, BM?-15, BMP-3b, DPP; Vg1; Vgr; 60Aprotein; GDF-1; GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10,GDF-11; and morphogenically active amino acid sequence variants thereof.118. The composition of claim 104 wherein said osteogenic protein isselected from the group consisting of: OP1; OP2, BMP2; BMP4; BMP5; BMP6;and morphogenically active amino acid sequence variants thereof. 119.The composition of claim 104 wherein said osteogenic protein is amorphogen, said morphogen comprising an amino acid sequence having atleast 70% homology within the C-terminal 102-106 amino acids, includingthe conserved seven cysteine domain, of human OP1.
 120. The compositionof claim 104 wherein said osteogenic protein is OP1.
 121. Thecomposition of claim 104 wherein said osteogenic protein is mature OP 1solubilized in a saline solution.
 122. The composition of claim 104wherein said osteogenic protein comprises an amino acid sequence definedby OPX (Seq. ID No.3); Generic Seq. 6 (Seq. ID No.4); Generic Seq. 7(Seq. ID No.5); Generic Seq. 8 (Seq. ID No.6); or Generic Seq. 9 (Seq.ID No.7).